Modulating the cellular stress response

ABSTRACT

Methods of using B2 or Alu nucleic acids, or antisense oligonucleotides that modulate the EZH2/B2 or EZH2/Alu interaction and have the capacity to alter cleavage of B2 and Alu RNA, for increasing or decreasing cell and organismal viability.

CLAIM OF PRIORITY

This application is a divisional of U.S. patent application Ser. No. 16/308,638, filed Dec. 10, 2018, which is a § 371 national stage application of International Application No. PCT/US2017/036829, filed on Jun. 9, 2017, which claims the benefit of U.S. Provisional Patent Application Serial Nos. 62/433,770, filed on Dec. 13, 2016; 62/408,639, filed on Oct. 14, 2016; and 62/347,737, filed on Jun. 9, 2016. The entire contents of the foregoing are hereby incorporated by reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant Nos. R01-GM090278 awarded by the National Institutes of Health and Zo 287/4-1 awarded by the German Research Foundation. The Government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 20, 2021, is named Sequence_Listing.txt and is 30,950 bytes in size.

TECHNICAL FIELD

Described herein are methods of using Alu or B2 nucleic acids, or antisense oligonucleotides that modulate the EZH2/B2 or EZH2/ALU interaction and have the capacity to alter cleavage of B2/ALU and its expression levels, for increasing or decreasing whole-organism or cell health, proliferation potential, functionality and viability, such as during various types of environmental stress (thermal (e.g., heat or cold), radiation, chemical, or hypoxic stress), inflammation, infection, and cancer.

BACKGROUND

Environmental stress is an everyday reality for all organisms. A rapid and effective response is essential for survival in the face of acute stress, such as those resulting from exposure to extreme temperatures (cold, heat), chemical toxin, radiation, and infection. Activation of the so-called stress response genes protects cells from conditions that would normally be lethal, and a failure to mount an effective or controlled stress response can lead to a variety of diseases, including cancer and autoimmunity. Cancer therapeutic agents often target components of the stress/heat shock response pathway to overcome unchecked growth of cancer cells, but cancer cells frequently respond by mutating these stress-control genes (Chircop and Speidel, 2014). A better understanding of how the stress response is controlled would therefore be beneficial towards human health.

SUMMARY

More than 98% of the mammalian genome is noncoding and interspersed transposable elements account for ˜50% of noncoding space. Because of their repetitive nature and relative lack of conservation, these elements have been termed “junk DNA”. As demonstrated herein, an interaction between the Polycomb protein, EZH2, and RNA made from B2 SINE retrotransposons controls the stress response. Using the heat shock model, the present results show that B2 RNA binds stress genes and suppresses their transcription before stress. Upon stress, EZH2 is recruited and triggers cleavage of B2 RNA. B2 degradation in turn upregulates stress genes. Evidence indicates that B2 RNA operates as “speed bumps” to slow progression of RNA polymerase and stress rapidly releases the brakes on transcription. Thus, the present inventors have attributed a new function to EZH2 that is independent of its histone methyltransferase activity and revealed that EZH2 and B2 together control the activation of a large network of stress-response genes. In humans, the B2 element is known as ALU. As shown herein, ALUs are also subject to cleavage.

Thus, provided herein are methods for of modulating health, proliferation potential, functionality or viability of a cell or tissue, comprising contacting the cell with an antisense oligonucleotide (ASO) comprising at least one locked nucleotide that binds to an Alu or B2 RNA and alters levels of the Alu or B2 RNA, by promoting or blocking cleavage of the B2/Alu RNA. As used herein, functionality means the typical physiological function of the cell, e.g., a pancreatic beta cell that is alive but not producing insulin is viable but not functional. Neural or muscle cells with an ion channel disorder are still viable but cannot transmit or receive the message, thus they are not functional.

In some embodiments, the cell is in a subject who suffers from an inflammatory or autoimmune disorder affecting the cell.

In some embodiments, the cell is in a subject who suffers from a degenerative disorder affecting the cell.

In some embodiments, the degenerative disorder is macular degeneration.

Also provided herein are methods for enhancing health or viability of a cell, comprising contacting the cell with an antisense oligonucleotide (ASO) comprising at least one locked nucleotide that binds to an Alu or B2 RNA and promotes cleavage of the Alu or B2 RNA, preferably wherein the ASO is an siRNA, shRNA or comprises at least one locked nucleotide, e.g., is a gapmer or mixmer.

In some embodiments, the cell is in a subject who suffers from an environmental stress.

In some embodiments, the environmental stress is infection, thermal (e.g., heat or cold), radiation, or chemical exposure or hypoxic stress.

Also provided herein are methods for promoting or inhibiting proliferation of a cell, comprising contacting the cell with an antisense oligonucleotide (ASO) that binds to an Alu or B2 RNA and reduces binding of EZH2 to the Alu or B2 RNA and inhibits or promotes cleavage of the Alu or B2 RNA.

Further provided herein are methods for promoting or inhibiting apoptosis in a cell, comprising contacting the cell with an antisense oligonucleotide (ASO) that binds to an Alu or B2 RNA and reduces binding of EZH2 to the Alu or B2 RNA and inhibits or promotes cleavage of the Alu or B2 RNA.

In some embodiments, proliferation is inhibited, or apoptosis is promoted, and the cell is a cancer cell. In some embodiments, the cancer cell is in a subject who has cancer; optionally, the ASO is administered locally to the cancer in the subject.

In some embodiments, the ASO is selected from the group consisting of peptide nucleic acids, N3′,P5′-phosphoramidates, morpholino phosphoroamidates, 2′-O-methoxyethyl nucleic acids, or ribonucleic acids delivered through an RNA degradation protective carrier.

Also provided herein are compositions comprising a plurality of isolated antisense oligonucleotides (ASOs), preferably each comprising at least one locked nucleotide, that target a plurality of different Alu or B2 sequences and mediate or promote cleavage of the sequences, and a pharmaceutically acceptable carrier.

Also provided herein are compositions for use in a method of promoting viability of a cell, preferably a cell in a living subject, comprising a plurality of isolated antisense oligonucleotides (ASOs), preferably each comprising at least one locked nucleotide, that target a plurality of different Alu or B2 sequences and mediate or promote cleavage of the sequences, and a pharmaceutically acceptable carrier In some embodiments, the subject suffers from an autoimmune disorder or a degenerative disorder.

Additionally, provided herein are compositions comprising a plurality of antisense oligonucleotides that target a plurality of different Alu or B2 sequences and inhibit cleavage of the sequences, and a pharmaceutically acceptable carrier.

In some embodiments, the ASO is selected from the group consisting of peptide nucleic acids, N3′,P5′-phosphoramidates, morpholino phosphoroamidates, 2′-O-methoxyethyl nucleic acids, and ribonucleic acids delivered through an RNA degradation protective carrier.

Further provided herein are compositions for use in a method of decreasing viability of a cell, comprising a plurality of antisense oligonucleotides that target a plurality of different Alu or B2 sequences and inhibit cleavage of the sequences, and a pharmaceutically acceptable carrier.

In some embodiments, the cell is a cancer cell in a subject.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A-F. B2 RNA associates with PRC2 and can be detected as multiple shorter species in vivo.

A) Relative B2 representation (red pie slice) among SINEs in the mouse genome, among the female ES cell transcriptome (RNA-seq), and among the EZH2 interactome (RIP-seq), as indicated. Right pie chart is reproduced from (Zhao et al., 2010) and depicts relative representation of SINEs among all reads in the PRC2 interactome.

B) Top panel: Distribution of EZH2 RIP-seq reads around the start site (+/−2000 bp) of two classes of SINE elements, B2 and B1. Repeats of each class have been collapsed into a metagene with a common start site. B2 RNA is enriched but B1 is not, in spite of their relatively equal expression levels in ES cells, as shown by RNA-seq (bottom two panels).

C) Distribution of EZH2 RIP-seq reads within the B2 element. Upper panel: Distribution of reads across a metagene profile inclusive of all B2 elements aligned to their start from nucleotides 1-201 (x-axis/absolute distance in nucleotides from repeat start is maintained in the metagene). Lower panel: Alignment of EZH2 RIP-seq reads within the B2 metagene. Sharp discontinuities implie existence of different B2 subfragments.

D) Distribution of short RNA-seq reads within the B2 element (upper panel) and alignment of these reads within the B2 metagene between nt 1-201 (lower panel).

E) Top panel: Map, structure, and critical domain of B2 RNA as determined previously (Espinoza et al., 2007); SEQ ID NO:73. Bottom panel: 5′ ends of the short RNA-seq reads are plotted along the B2 locus (x-axis). Red X's (Top panel) and asterisks (Bottom panel) mark sites of discontinuity, as observed by the short RNA-seq analysis.

F) Top left: Binding isotherms of EZH2 generated from data obtained from double-filter binding experiments. Top right: Table of K_(d) and R² values for EZH2-B2 RNA interactions. Bottom: Filter binding assay performed as previously described (Cifuentes-Rojas et al., 2014) for B2 RNA and EZH2. RepA I-IV and RepA I-II were used as positive controls and MBP and P4P6 as negative controls. Error bars within binding curves and standard deviations (SD) within the table represent three independent experiments. U, unbound; B, bound.

FIGS. 2A-K. EZH2 triggers cleavage of B2 RNA in vitro.

A) B2 sub-family consensus sequences of the 5′ end, inclusive of the TSS, Box A and B motifs, and the major site of discontinuity at position 98 for B3 (SEQ ID NO:65), B2_Mm1a (SEQ ID NO:66), B2_Mm1t (SEQ ID NO:67), and M2_Mm2 (SEQ ID NO:68).

B) Incubation of in vitro-transcribed B2 RNA (200 nM) with purified recombinant EZH2 (25 nM) results in B2 cleavage and loss in vitro after 13 hours at 22° C. in vitro. Arrowhead, full-length B2 RNA. Asterisks, cleaved B2 fragments.

C) Incubation with 25 nM purified control proteins, GST and EED, does not result in significant cutting after 13 hours at 22° C. in vitro. Arrowhead, full-length B2 RNA. Asterisks, cleaved B2 fragments.

D) Cleaved RNA fragments (asterisks) are purified, adapter ligated, reverse-transcribed, and subjected to deep sequencing. Start coordinates for the sequenced reads are mapped along the x-axis. Arrowhead, full-length B2 RNA.

E) Incubation of in vitro-transcribed RNAs (100 nM) with purified recombinant EZH2 (50 nM) results in cleavage only of B2 RNA. RNAs were mixed with EZH2 and incubated at 37° C. or 4° C. for 30 min. B2 was also incubated with FLAG peptide (50 nM) at 37° C. as control.

F) Kinetic analysis of B2 cleavage in the presence of EZH2 protein. 25 nM EZH2 was incubated with 200 nM B2 RNA at 37° C. for 0-100 minutes and the products were run on a 6% TBE-Urea-PAGE. Arrowhead, full-length B2 RNA. Asterisks, cleaved B2 fragments.

G) Fraction of full-length B2 RNA at each time point from panel E (arrow) was plotted as a function of time. Cleavage rate constants were then determined by a linear fit using the differential form of the rate equation for an irreversible, first-order reaction. The slope is the observed cleavage rate constant (k_(obs)). R² values indicate that data points have an excellent fit to the curve. Two independent experiments have been used for this plotting.

H) Table of calculated ob k_(obs) and RNA half-lives for B2 in the presence of various test proteins.

I) Rate of B2 cleavage depends on the concentration of EZH2 protein. 50 nM B2 RNA is incubated with increasing concentrations of EZH2 for 20 minutes at 37° C. in vitro. The products were then run on a 6% TBE-Urea PAGE. Arrowhead, full-length B2 RNA. Asterisks, cleaved B2 fragments.

J) Kinetic analysis showing that the rate of B2 cleavage depends on the concentration of EZH2. 200 nM B2 RNA is incubated with increasing EZH2 concentrations (25-500 nM) at 37° C. and the amount of remaining full-length B2 RNA is plotted as a function of time. Cleavage rate constants were then determined by a linear fit using the differential form of the rate equation for an irreversible, first-order reaction. The slope approximated observed rate constant (k_(obs)). R² values indicate that datapoints have an excellent fit to the curve. Two independent experiments have been used for this plotting.

K) k_(obs) values from panel I are plotted as a function of EZH2 concentration. High R² values indicate that data points have an excellent fit to the curve.

FIGS. 3A-D. Heat shock destabilizes B2 RNA in vivo.

A) Full-length B2 RNA was pre-incubated with 25 nM EZH2 for 7 h at 37° C. The RNA was then gel purified and either the whole B2 or the subfragments were then transfected into NIH/3T3 cells and cells were grown at 37° C. Mock represents transfection without any RNA. Photographs were taken after 3 days.

B) NIH/3T3 cells transfected with either synthesized full-length B2 RNA or a synthesized B2 fragment starting at position 99. Cells were then allowed to recover for 2-5 days. Cell were photographed (left panels) and counted (right panels) at days 2 and 5.

C) Diagram of the heat shock response. Hundreds of genes are increased in expression (“upregulated”), and others are decreased in expression (“downregulated”). B2 expression increases within 15 minutes of heat shock.

D) Short RNA-seq of NIH/3T3 cells before and after heat shock (45° C. for 15 minutes). Two biological replicates yielded similar results. 5′ ends of short RNA-seq reads are mapped to the B2 transcript and the relative number of 5′ ends is plotted on the y-axis. The 5′ end counts are normalized to the number of full length B2 RNAs to account for any possible changes in the general B2 levels during heat shock (KS test; P<0.0001).

FIGS. 4A-G. CHART-seq analysis: B2 RNA binds heat shock responsive genes in vivo.

A) For CHART-seq analysis, a cocktail of 17-base B2 capture probes is designed to span nt 87-103 and overlap the major cut site. Thus, the cocktail should only pull down chromatin regions associated with full-length B2 RNA. The cocktail contains a pool of oligos that would capture SNP variants for the vast majority of B2 elements.

B) Genome-wide peak annotation analysis (Galaxy) of the distribution of B2 CHART peaks with reference to UCSC RefSeq genes.

C) Pie charts (PAVIS) showing relative distributions of B2 CHART hits genome-wide with reference to different mm9 RefSeq gene features. A comparison of the relative genomic representation for each feature is shown in the bottom pie chart. Satellites represent 0.1% of the total and in this resolution are not visible.

D) An exon/intron 1-focused metagene analysis of B2 CHART reads shows a significant decrease of B2 binding within intron 1 after heat shock (KS test, P<0.0001).

E) IGV screenshots of B2 binding patterns for two H/S-upregulated and two H/S-downregulated genes, along with RNA-seq data. Pre- and post-H/S profiles are shown. Paired data are shown at the same scale (numbers in brackets, right) for comparison.

F) B2 binding across TSS-centered metagene profiles+/−1000 bp of flanking sequence. Pre- and post-H/S traces are shown for all genes, upregulated genes (Table 1), and downregulated genes (Table 2), as indicated. Analysis from two biological replicates corresponds to an FDR<0.05 estimation of noise to input signal, and an E-value of 1000. Statistical significance (P) of the difference between pre- and post-H/S read counts is determined by KS test (P<0.0001).

G) Relative change in B2 binding after H/S. Relative change is indicated by the ratio of post- to pre-H/S CHART reads as described in methods. Positive and negative values represent an increase and decrease in B2 binding after heat shock, respectively. The metagene profiles are centered on the TSS of up- and down-regulated genes, as indicated (KS test, P<0.0001) for the read distribution changes between up- and down-regulated genes).

FIGS. 5A-F. Loss of B2 binding induces H/S-responsive genes.

A) Metagene analysis of changes in POL-II-S2P binding (ChIP-seq) at H/S-upregulated and -downregulated genes. Analysis corresponds two biological replicates and an FDR<0.05 estimation of noise to input signal. Statistical significance (P) between pre- and post-H/S read counts is determined by KS test (P<0.0001).

B) Metagene analysis of changes in POL-II-S2P binding at Type I (B2 binding in pre-H/S) and Type II (B2 binding in post-H/S) genes. Analysis performed as in (A) (KS test, P<0.0001).

C) Metagene analysis showing relative changes in POL-II-S2P binding after H/S for Types I and II genes. Relative change is indicated by the ratio of post- to pre-H/S ChIP coverage. Positive and negative values represent an increase and decrease in POL-II-S2P density, respectively (KS test (P<0.0001) for the read distribution changes between Type I and Type II genes).

D) Cleavage of B2 RNA induced by B2-specific LNA. NIH/3T3 cells are transfected with B2 or Scr LNAs and short RNA-seq analysis is performed after 24 hours. 5′ ends of short RNA-seq reads are mapped to the B2 transcript and the relative number of 5′ ends is plotted on the y-axis (KS test, P<0.0001)

E) ChIP-seq analysis indicates that B2 LNA recapitulates increased POL-II-S2P density across H/S-upregulated genes without application of heat shock (KS test, P<0.0001).

F) Metagene analysis of RNA-seq data demonstrates that B2 LNA treatment also recapitulates increased expression of H/S-upregulated genes in the absence of H/S (KS test, P<0.0001).

FIGS. 6A-J. EZH2 is recruited to B2 target genes to direct H/S activation.

A) Metagene analysis of changes in EZH2 binding (ChIP-seq) at H/S-upregulated and -downregulated genes. Analysis corresponds to two biological replicates (FDR<0.05 for sample signal to input noise) and P<0.0001 (KS test) between pre- and post-H/S read count distribution of downregulated genes only.

B) EZH2 is recruited to H/S-responsive genes with a B2-binding site. Metagene analysis of changes in EZH2 binding (ChIP-seq) at H/S-upregulated with or without B2 binding sites (Type I versus Type II). P<0.0001 (KS test) for upregulated genes with B2 binding site.

C) H3K27me3 coverage is not increased at the TSS after EZH2 recruitment to H/S-upregulated genes. The metagene analysis is performed on the subclass of H/S-upregulated genes with B2 and EZH2 binding sites (either before or after H/S) (Difference not statistically significant, KS test).

D) Metagene analysis showing relative changes in H3K27me3 coverage after H/S for the subclass of upregulated genes shown in (C). Relative change is indicated by the ratio of post- to pre-H/S ChIP coverage. Positive and negative values represent an increase and decrease in H3K27me3 coverage, respectively.

E) Meta-site analysis centered on the EZH2 binding site shows B2 binds in pre-H/S cells where EZH2 is gained after H/S. x=0 corresponds to EZH2 peaks start of post-H/S cells.

F) Meta-site analysis centered on the B2 binding site shows that EZH2 binds where B2 is lost during H/S. x=0 corresponds to B2 peaks of pre-H/S cells.

G) Anti-correlation of B2 and EZH2 binding viewed in a metagene plot. Relative changes in either B2 or EZH2 coverage at upregulated genes are shown after H/S. Relative change is indicated by the ratio of post- to pre-H/S coverage. Positive and negative values represent an increase and decrease in density, respectively.

H) Linear anti-correlation between B2 coverage and EZH2 density. Change in B2 density (x-axis) plotted as a function of change in EZH2 density (y-axis). R=−0.7, P<0.05.

I) Depleting EZH2 reduces processing of B2 RNA. NIH/3T3 cells are transfected with EZH2 or Scr LNAs and short RNA-seq analysis is performed after 24 hours. 5′ ends of short RNA-seq reads are mapped to the B2 transcript and the relative number of 5′ ends is plotted on the y-axis (P<0.0001, KS test).

J) EZH2 is required for the heat shock response. Metagene analysis of RNA-seq data demonstrates that EZH2 depletion reduces expression of H/S-upregulated genes (P<0.0001, KS test for pre-post-HS distributions).

FIGS. 7A-B. The Speed Bump Model of B2/EZH2-mediated gene control.

A) Compilation of data from FIGS. 4-6: IGV screenshots showing alignments of binding patterns for B2 RNA, EZH2, and POL-II-S2P to specific genes.

B) The Speed Bump Model. Upper panels: In resting cells, B2 RNA binds H/S-responsive genes and reduces their expression by establishing “speed bumps” for POL-II progression. Upon stress (e.g., heat shock), PRC2 is recruited to H/S-responsive genes and triggers B2 degradation. The speed bumps are removed and POL-II elongates at faster speed, thereby resulting in transcriptional upregulation. Bottom panels: B2 also regulates housekeeping genes that undergo transcriptional downregulation upon H/S. H/S results in B2 upregulation. These newly transcribed B2 RNA binds new target genes and reduces POL-II activity, thereby reducing expression of housekeeping genes. Both transcriptional initiation and elongation may be affected. The speed bump mechanism enables a rapid and specific response to cellular stress. All changes are observed within 15 minutes of heat shock.

FIG. 8. Correlation between biological replicates of the B2 CHART-seq experiment.

Metagene plot of B2 CHART read density at B2 elements, the site of nascent transcription. As expected, B2 RNA is enriched at the site of transcription. These loci served as positive control and are excluded from further analysis.

FIG. 9. Correlation between biological replicates of RNA-seq data after EZH2 knockdown.

Significant EZH2 knockdown by LNA transfection. P=0.04, as determined by t-test.

FIGS. 10A-C. Human Alu are the equivalent of mouse B2, and are also cleaved.

A) Human Alu consensus sequence (SEQ ID NO:1) and secondary structure adapted from Hadjiargyrou and Delihas, Int J Mol Sci. 14(7):13307-28 (2013). Alu sequence consists of a sequence dimer, of which the monomers constitute its left and right arms, respectively. The asterisk indicates the Alu cut point in vivo as defined in FIG. 10B below.

B) Alu's are cut at a position within the position range 49-52 from the start of the Alu SINE genomic elements. The graph shows 5′ ends of short RNA-seq reads mapped against mm9 genomic Alu elements creating the transcript metagene of the Alu elements. The metagene x axis is constructed by aligning the 5′ end start points of all Alu RNAs as defined in UCSC repeat masker as of September 2016. The x axis position numbers represent absolute distance in nucleotides from the Alu start site (i.e. position 1 in the metagene corresponds to the start site of each Alu genomic element from which the Alu RNA transcript metagene is constructed). The relative number of short RNA 5′ ends is plotted on the y-axis. Because, as shown in Table 3, various Alu elements present variations from the consensus sequence showed in FIG. 10A, the cut position varies accordingly based on various insertions and deletions of each Alu that constitutes this metagene (i.e. cut position of different Alu sabfamilies relative to the Alu start site is heterogenous based on these variations creating the compound metagene profile of this figure). These variations and the respective cut range (highlighted in gray), are shown in FIG. 11. Mapping is focused on only the first Alu Arm (left) to prevent cross mapping because of sequence similarity between the two Alu sequence dimers.

C) For a specific Alu class, AluY, the cut is at position 51. This is presented as an example of the cut point within an Alu subfamily.

FIG. 11. Table of sequences for human Alu family members and their respective cut sites. Each row represents the sequence of an Alu family aligned with each other based on Vassetzky amd Kramerov, Nucleic Acids Res. 41(Database issue):D83-9 (2013). The cut region is highlighted in grey. These sequences represent the consensus sequences of all human Alu subfamilies.

DETAILED DESCRIPTION

For more than half a century, genome size has been known to correlate poorly with organism size and developmental complexity (Gall, 1981; Mirsky, 1951; Thomas, 1971). Many flowering plants and amphibians, for example, have genome sizes (or C-value) that are 10- to 100-times larger than those of mammals. This so-called “C-value paradox” was thought to be solved by the discovery that only 1-2% of mammalian genomes have protein-coding potential. The rest of the genome consists largely of repetitive DNA, with satellite DNA, retrotransposable elements, and DNA transposons accounting for ˜50% of noncoding sequences (de Koning et al., 2011). For much of the past few decades, these poorly conserved elements have been considered “junk DNA”, believed to be remnants of evolution and genetic parasites that proliferate without constraint of purifying selection (Kramerov and Vassetzky, 2011). Emerging studies, however, have been hinting at possible functions for these noncoding sequences (Bourque et al., 2008; Lowe and Haussler, 2012; Lunyak et al., 2007; Ponicsan et al., 2010). It is now known through ENCODE that >80% of the noncoding genome is transcribed during development (Consortium et al., 2007). A growing number of the resulting long noncoding RNAs (lncRNA)—particularly the unique ones—now appear to have important cellular roles, including during X-chromosome inactivation, genomic imprinting, and cancer progression (Kapranov et al., 2007; Lee and Bartolomei, 2013; Li et al., 2016; Rinn and Chang, 2012; Tay et al., 2014).

Nevertheless, functions for repetitive elements remain largely a mystery. One class of repeat elements, however, has garnered some attention in recent years. The B2 element belongs to a family of short intersperse nuclear element (SINE), is present in 100,000 copies, and is transcribed by RNA polymerase III into a 180- to 200-base lncRNA (Kramerov et al., 1982; Kramerov and Vassetzky, 2011) with a 5′ tRNA-like sequence and A-rich 3′ end (Daniels and Deininger, 1985; Krayev et al., 1982; Lawrence et al., 1985). B2 expression changes significantly during development (Bachvarova, 1988) and its expression is highly induced by specific cellular stresses and disease states, such as viral infection (Singh et al., 1985), age-related macular degeneration (Kaneko et al., 2011; Tarallo et al., 2012), and various cancers (Kaczkowski et al., 2016; Kramerov et al., 1982; Moolhuijzen et al., 2010). The functional and mechanistic relationships between B2 and these various disease states are not currently known. Notably, B2 RNA has been shown to play a role in heat shock (Fornace and Mitchell, 1986; Li et al., 1999), during which B2 RNA is assembled into the pre-initiation complex of RNA polymerase II (POL-II) (Espinoza et al., 2004) and becomes inhibitory to transcription in vitro (Allen et al., 2004). Transcription of the B2 element has also been implicated in formation of a boundary between heterochromatin and euchromatin (Lunyak et al., 2007). The B2 DNA element can also lend its promoter activity to mammalian genes (Ferrigno et al., 2001). Thus, in the mammalian noncoding genome, the B2 repeat currently stands out as one element that is likely to be much more than junk.

With this in mind, we became intrigued by a set of data involving the RNA-binding activity of an epigenetic complex known as Polycomb repressive complex 2 (PRC2)(Zhao et al., 2010). PRC2 is a histone methyltransferase complex consisting of four core subunits, EED, RBBP4/7, SUZ12, and the catalytic subunit EZH2, that together mediate the trimethylation of histone H3 at lysine 27 (H3K27me3) and help to establish repressive chromatin (Margueron and Reinberg, 2011). By RNA immunoprecipitation with deep sequencing (RIP-seq), previous work in mouse cells revealed an RNA interactome of >9,000 unique transcripts (Zhao et al., 2010). While the raison d'etre for the large RNA interactome is under intensive investigation (Cifuentes-Rojas et al., 2014; Davidovich et al., 2015; Davidovich et al., 2013; Kaneko et al., 2013), it is clear that interacting transcripts can target PRC2 in cis to repress gene expression (Pandey et al., 2008; Zhao et al., 2010; Zhao et al., 2008). Further examination of PRC2-RNA interactions has also shown that PRC2 binding can be found at active genes (Davidovich et al., 2013; Kaneko et al., 2013), implying that PRC2 may not solely be involved in gene repression.

The PRC2 RIP-seq analysis also identified RNAs made from repetitive elements (Zhao et al., 2010). However, because repeats pose technical challenges for sequence alignment during analysis of next-generation sequencing data (Treangen and Salzberg, 2012), the repeat fraction had been unexamined despite the fact that such transcripts were present in large numbers. Described herein is an exploration of PRC2's interaction with repetitive RNAs. These findings integrate two previously unconnected networks—Polycomb and junk RNA—in the cellular response to stress and demonstrate the importance of a B2-specific RNA cleavage event. Herein, data show that EZH2 and a B2 transcript made from “junk” DNA play a central role (FIG. 7B). Intriguingly, the key triggering event is B2 RNA elimination. Without wishing to be bound by theory, it is proposed that B2 RNA act as transcriptional “speed bumps” for POL-II. B2 RNA binds broadly in intronic regions, sometimes to one intron, sometimes to two or more (FIG. 4B-E, 7A). The present data suggest that, in resting cells, B2 binding to gene bodies reduces the elongation rate of POL-II and thereby controls the rate at which target genes are expressed in the unstressed state.

Upon stress, EZH2 is rapidly recruited to H/S-responsive genes (within 15 minutes). A significant consequence is a degradation of B2 RNA involving endonucleolytic cleavages at multiple positions (e.g., nt 98, 77, 33) both in vitro and in vivo (FIG. 1E, 2D, 3D, 61). Cleavage of B2 RNA is sufficient to induce H/S-responsive genes (FIG. 5E, F, 7A). Notably, cut B2 fragments have dramatically reduced affinities for EZH2 (ΔK_(d) from 423 nM to >3000 nM; FIG. 1F). Without wishing to be bound by theory, it is suggested that the cleavage event results in disintegration and release of B2 RNA from target genes. B2 degradation at target genes removes the POL-II speed bumps, enabling a larger percent of elongating POL-II to reach the 3′ termini of target genes. Previous studies had shown transcriptional pausing downstream of H/S-responsive promoters (Brown et al., 1996; Kwak et al., 2013). Speculatively, some pause sites may correspond to sites of B2 binding. A B2 speed bump mechanism would enable a swift cellular response to stress, as EZH2 recruitment and B2 cleavage occur rapidly—within minutes of the stimulus in vivo.

The present study ascribes a specific new function to EZH2 that is independent of its well-known histone methyltransferase activity. Although this work was conducted in mammalian cells, EZH2 may also function during stress in flies, plants, and fungi, (Basenko et al., 2015; Kleinmanns and Schubert, 2014; Siebold et al., 2010). The present work also provides an explanation for the paradoxical observation that EZH2 and its associated RNAs can be found at both active and inactive genes (Davidovich et al., 2013; Kaneko et al., 2013; Zhao et al., 2010). Whereas the H3K27me3 mark is a critical part of EZH2-mediated gene silencing (Margueron and Reinberg, 2011), gene activation by the EZH2-B2 interaction does not depend on H3K27 trimethylation (FIG. 6C,D). Rather activation depends on contact-dependent B2 elimination. Thus, frequent mutation of EZH2 (Margueron and Reinberg, 2011) and misexpression of Alu/B2 elements (Chircop and Speidel, 2014; Kaczkowski et al., 2016; Kramerov et al., 1982; Moolhuijzen et al., 2010) in cancer cells may in part be explained by the critical roles played by EZH2 and B2 during the stress response. Finally, it should be noted that heat shock normally leads to two distinct responses—transcriptional upregulation of stress response genes (Table 1) and transcriptional downregulation of housekeeping genes, among others (Table 2). The EZH2-B2 dynamic relates primarily to the former set of genes. B2 plays an equally important role for the latter (FIG. 3C, 7B). Repression of a large number of genes that are non-essential to stress is an adaptation to conserve cellular resources. Existing studies have demonstrated a role for B2 RNA in repression of two housekeeping genes, including ActinB and Hk2 (Allen et al., 2004; Espinoza et al., 2004; Fornace and Mitchell, 1986; Li et al., 1999). The B2 CHART-seq data now provide a genomic view for this second arm of the heat shock response and reveal that a large number of genes are targeted by B2 RNA immediately after heat shock (FIG. 4F,G; Tables S2,S4,S7), concurrently with the increase in B2 expression (Allen et al., 2004; Fornace and Mitchell, 1986). Because EZH2 is not recruited to the downregulated gene set, B2 RNA is spared the degradation. Previous studies convincingly showed that incorporated B2 can act in vitro by blocking formation of the POL-II pre-initiation complex at promoters. The present findings suggest that B2 may suppress both transcriptional initiation and elongation in vivo. Notably, the present study explains how H/S-upregulated genes can be immune to increased B2 expression immediately following heat shock, as indeed the recruitment of EZH2 ensures B2 degradation at H/S-upregulated genes. In conclusion, the present results have shown that a specific interaction between EZH2 and B2 “junk RNA” triggers the heat shock response via an RNA elimination event.

Methods of Modulating the Mammalian Stress Response

The present results demonstrate that EZH2 interaction with B2 SINE retrotransposons triggers PRC2-mediated cleavage of the B2 elements (consensus sequences are shown in FIG. 2A; the ASOs targeting B2 included a mixture of 5′-GTTACGGATGGTTGTG-3′ (SEQ ID NO:63) and 5′-TGTAGCTGTCTTCAG-3′ (SEQ ID NO:64) LNAs, e.g., the +in front of the base depicts an LNA nt: 5-G+TTA+CGG+ATGG+TTG+TG-3 (SEQ ID NO:69) and 5-TG+T+AGC+TGTC+TTC+AG-3′ (SEQ ID NO:70)), inducing the heat shock response in mammalian cells. Antisense oligonucleotides that modulate the EZH2/B2 interaction have the capacity to alter cleavage of B2. Non-cleaving antisense oligos (ASOs) that prevent or decrease binding of EZH2 to B2 without increasing cleavage of B2 can increase levels of intact B2, resulting in cell death. Such pro-apoptotic ASOs would be useful, e.g., in conditions associated with unwanted cellular proliferation, such as cancer. These ASOs include peptide nucleic acids, N3′,P5′-phosphoramidates, morpholino phosphoroamidates, 2′-O-methoxyethyl nucleic acids, and ribonucleic acids delivered through an RNA degradation protective carrier (e.g., using the the HiPerfect reagent from Qiagen; see, e.g., Zovoilis et al., EMBO J. 2011 Sep. 23; 30(20):4299-308). This includes sequences that have both continuing stretches of the modification or clusters of modified nucleotides separated by not modified ones.

In contrast, ASOs such as Locked Nucleic Acids (LNAs, ribonucleotides containing a “lock” or methylene bridge that connects the 2′-oxygen of ribose with the 4′-carbon), that increase cleavage of B2 elements (e.g., by RNAseH) would increase cell viability, useful in conditions associated with cell death such as autoimmune diseases, degenerative diseases, and ischemic injury. Cyclohexenyl nucleic acids can also be used. See, e.g., Kurreck et al., Nucleic Acids Res. 30(9): 1911-1918 (2002). Also as shown herein, the introduction of B2 RNA into a cell, e.g., a cancer cell, induces cell death. Thus the present methods can include administration of an Alu or B2 RNA, or a DNA encoding an Alu or B2 RNA, or a fragment thereof (RNA or DNA), to induce cell death.

Human Alu Repeats

Repetitive DNA elements account for at least about 20% of the human genome, and have been classified into four principal families of interspersed repeats; Alu, Line 1, MIR and MaLR (Schmid, Prog. Nucleic Acid Res. Mol. Biol., 53:283-319 (1996)). The rodent B2 family of repetitive sequence elements corresponds to the human Alu sequence family (see, e.g., Clawson et al., Cell Growth and Diff 7(5):635-646 (1996)); thus, in the methods described herein, Alu sequences can be used as a target for modulating the stress response in humans. The Alu sequences are typically about 280-300 nucleotides in length, and account for about 11% of the human genome (Lander et al., Nature, 409, 860-921 (2001); Deininger et al., Genome Biol. 2011; 12(12): 236). Exemplary consensus sequences of human Alu repeats can be found in FIG. 11; see also FIG. 1 of Weisenberger et al., Nucleic Acids Research 33(21):6823-36 (2005); in FIG. 1 of Luo et al., Biomed Res Int. 2014:784706 (2014); and in Hambor et al., Molecular and Cellular Biology, 13(11): 7056-7070 (1993).

Antisense Oligonucleotides (ASOs)

In some embodiments, the ASOs used in the present methods are 10 to 50, 10 to 20, 10 to 25, 13 to 50, or 13 to 30 nucleotides in length. One having ordinary skill in the art will appreciate that this embodies ASOs having complementary portions of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length, or any range therewithin. In some embodiments, the ASOs are 15 nucleotides in length. In some embodiments, the ASOs are 12 or 13 to 20, 25, or 30 nucleotides in length. One having ordinary skill in the art will appreciate that this embodies ASOs having complementary portions of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length, or any range therewithin (complementary portions refers to those portions of the ASOs that are complementary to the target sequence). (As used herein, the “target sequence” or “target RNA” means B2 RNA, or Alu RNA in humans, or other equivalent sequences in other organisms).

The ASOs useful in the present methods are sufficiently complementary to the target RNA, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect. “Complementary” refers to the capacity for pairing, through hydrogen bonding, between two sequences comprising naturally or non-naturally occurring bases or analogs thereof. For example, if a base at one position of an ASO is capable of hydrogen bonding with a base at the corresponding position of a RNA, then the bases are considered to be complementary to each other at that position. 100% complementarity is preferred but not required.

Routine methods can be used to design an ASO that binds to the target sequence with sufficient specificity. In some embodiments, the methods include using bioinformatics methods known in the art to identify regions of secondary structure, e.g., one, two, or more stem-loop structures, or pseudoknots, and selecting those regions to target with an ASO. For example, “gene walk” methods can be used to optimize the inhibitory activity of the nucleic acid; for example, a series of oligonucleotides of 10-30 nucleotides spanning the length of a target RNA can be prepared, followed by testing for activity. Optionally, gaps, e.g., of 5-10 nucleotides or more, can be left between the target sequences to reduce the number of oligonucleotides synthesized and tested. GC content is preferably between about 30-60%. Contiguous runs of three or more Gs or Cs should be avoided where possible (for example, it may not be possible with very short (e.g., about 9-10 nt) oligonucleotides).

In some embodiments, the ASO molecules can be designed to target a specific region of the RNA sequence. For example, a specific functional region can be targeted, e.g., a region comprising a known RNA localization motif (i.e., a region in which EZH2 binds to the target nucleic acid, e.g., the region between position 70 and 160 at the sequences of the B2 mm1a sequence of FIG. 2A). Alternatively, or in addition, highly conserved regions can be targeted, e.g., regions identified by aligning sequences from disparate species such as primate (e.g., human) and rodent (e.g., mouse) and looking for regions with high degrees of identity. Percent identity can be determined routinely using basic local alignment search tools (BLAST programs) (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656), e.g., using the default parameters.

Once one or more target regions, segments or sites have been identified, e.g., within a sequence known in the art or provided herein, ASO compounds are chosen that are sufficiently complementary to the target, i.e., that hybridize sufficiently well and with sufficient specificity (i.e., do not substantially bind to other non-target RNAs), to give the desired effect.

In the context of this invention, hybridization means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. Complementary, as used herein, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a RNA molecule, then the ASO and the RNA are considered to be complementary to each other at that position. The ASOs and the RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, “specifically hybridisable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the ASO and the RNA target. For example, if a base at one position of an ASO is capable of hydrogen bonding with a base at the corresponding position of a RNA, then the bases are considered to be complementary to each other at that position. 100% complementarity is not required.

As noted above, a complementary nucleic acid sequence need not be 100% complementary to that of its target nucleic acid to be specifically hybridisable. A complementary nucleic acid sequence for purposes of the present methods is specifically hybridisable when binding of the sequence to the target RNA molecule interferes with the normal function of the target RNA to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the sequence to non-target RNA sequences under conditions in which specific binding is desired, e.g., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed under suitable conditions of stringency. For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C., more preferably of at least about 37° C., and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred embodiment, hybridization will occur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C., more preferably of at least about 42° C., and even more preferably of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

In general, the ASOs useful in the methods described herein have at least 80% sequence complementarity to a target region within the target nucleic acid, e.g., 90%, 95%, or 100% sequence complementarity to the target region within an RNA. For example, an antisense compound in which 18 of 20 nucleobases of the antisense oligonucleotide are complementary, and would therefore specifically hybridize, to a target region would represent 90 percent complementarity. Percent complementarity of an ASO with a region of a target nucleic acid can be determined routinely using basic local alignment search tools (BLAST programs) (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656). ASOs that hybridize to an RNA can be identified through routine experimentation. In general, the ASOs must retain specificity for their target, i.e., must not directly bind to, or directly significantly affect levels or expression levels of, transcripts other than the intended target.

For further disclosure regarding ASOs, please see US2010/0317718 (antisense oligos); US2009/0181914 and US2010/0234451 (LNAs); and WO2010/129746 and WO2010/040112 (ASOs), as well as WO 2012/065143, WO 2012/087983, and WO 2014/025887 (ASOs targeting non-coding RNAs/supRNAs), all of which are incorporated herein by reference in their entirety.

In some embodiments, the ASOs used in the methods described herein are modified, e.g., comprise one or more modified bonds or bases. A number of modified bases include phosphorothioate, methylphosphonate, peptide nucleic acids, or locked nucleic acid (LNA) molecules. Some ASOs are fully modified, while others are chimeric and contain two or more chemically distinct regions, each made up of at least one nucleotide. These ASOs typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the target) and a region that is a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Chimeric ASOs of the invention may be formed as composite structures of two or more types of oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers (e.g., wherein a central block of DNA monomers is flanked by 2′-O modified ribonucleotides or other artificially modified ribonucleotide monomers such as bridged nucleic acids (BNAs), e.g., LNA/DNA/LNA or BNA/DNA/DNA gapmers, usually wherein the central block of deoxynucleotide monomers is sufficiently long to induce RNase H cleavage) or mixmers, i.e., LNAs containing a limited number of modified ribonucleotide or nucleotide monomers, e.g., LNA monomers, in combination with other types of monomers, typically DNA. See Wahlestedt et al., Proc. Natl Acad. Sci. USA, 97, 5633-5638 (2000). Representative United States patents that teach the preparation of such hybrid structures comprise, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of which is herein incorporated by reference.

In some embodiments, the ASO comprises at least one nucleotide modified at the 2′ position of the sugar, most preferably a 2′-O-alkyl, 2′-O-alkyl-O-alkyl or 2′-fluoro-modified nucleotide. In other preferred embodiments, RNA modifications include 2′-fluoro, 2′-amino and 2′ O-methyl modifications on the ribose of pyrimidines, abasic residues or an inverted base at the 3′ end of the RNA. Such modifications are routinely incorporated into oligonucleotides and these oligonucleotides have been shown to have a higher Tm (i.e., higher target binding affinity) than; 2′-deoxyoligonucleotides against a given target.

A number of nucleotide and nucleoside modifications have been shown to make the ASO into which they are incorporated more resistant to nuclease digestion than the native oligodeoxynucleotide; these modified oligos survive intact for a longer time than unmodified ASOs. Specific examples of modified ASOs include those comprising modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Most preferred are ASOs with phosphorothioate backbones and those with heteroatom backbones, particularly CH2-NH—O—CH2, CH, ˜N(CH3)˜O˜CH2 (known as a methylene(methylimino) or MMI backbone], CH2-O—N(CH3)-CH2, CH2-N(CH3)-N(CH3)-CH2 and O—N(CH3)-CH2-CH2 backbones, wherein the native phosphodiester backbone is represented as O—P—O—CH); amide backbones (see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374); morpholino backbone structures (see Summerton and Weller, U.S. Pat. No. 5,034,506); peptide nucleic acid (PNA) backbone (wherein the phosphodiester backbone of the ASO is replaced with a polyamide backbone, the nucleotides being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone, see Nielsen et al., Science 1991, 254, 1497). Phosphorus-containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3′alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′; see U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455, 233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563, 253; 5,571,799; 5,587,361; and 5,625,050.

Morpholino-based oligomeric compounds are described in Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510); Genesis, volume 30, issue 3, 2001; Heasman, J., Dev. Biol., 2002, 243, 209-214; Nasevicius et al., Nat. Genet., 2000, 26, 216-220; Lacerra et al., Proc. Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Pat. No. 5,034,506, issued Jul. 23, 1991.

Cyclohexenyl nucleic acid ASO mimetics are described in Wang et al., J. Am. Chem. Soc., 2000, 122, 8595-8602.

Modified ASO backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These comprise those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts; see U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264, 562; 5, 264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is herein incorporated by reference.

One or more substituted sugar moieties can also be included, e.g., one of the following at the 2′ position: OH, SH, SCH3, F, OCN, OCH3 OCH3, OCH3 O(CH2)n CH3, O(CH2)n NH2 or O(CH2)n CH3 where n is from 1 to about 10; Ci to C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF3; OCF3; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH3; SO2 CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an ASO; or a group for improving the pharmacodynamic properties of an ASO and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy [2′-0-CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl)] (Martin et al, Hely. Chim. Acta, 1995, 78, 486). Other preferred modifications include 2′-methoxy (2′-O—CH3), 2′-propoxy (2′-OCH₂ CH₂CH₃) and 2′-fluoro (2′-F). Similar modifications may also be made at other positions on the ASO, particularly the 3′ position of the sugar on the 3′ terminal nucleotide and the 5′ position of 5′ terminal nucleotide. ASOs may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.

ASOs can also include, additionally or alternatively, nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U). Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2′ deoxycytosine and often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, as well as synthetic nucleobases, e.g., 2-aminoadenine, 2-(methylamino)adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl)adenine and 2,6-diaminopurine. Kornberg, A., DNA Replication, W. H. Freeman & Co., San Francisco, 1980, pp 75-77; Gebeyehu, G., et al. Nucl. Acids Res. 1987, 15:4513). A “universal” base known in the art, e.g., inosine, can also be included. 5-Me-C substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2<0>C. (Sanghvi, Y. S., in Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions.

It is not necessary for all positions in a given ASO to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single ASO or even at within a single nucleoside within an ASO.

In some embodiments, both a sugar and an internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an ASO mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an ASO is replaced with an amide containing backbone, for example, an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds comprise, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al, Science, 1991, 254, 1497-1500. ASOs can also include one or more nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases comprise the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases comprise other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylquanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

Further, nucleobases comprise those disclosed in U.S. Pat. No. 3,687,808, those disclosed in ‘The Concise Encyclopedia of Polymer Science And Engineering’, pages 858-859, Kroschwitz, J.I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandle Chemie, International Edition′, 1991, 30, page 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications′, pages 289-302, Crooke, S. T. and Lebleu, B. ea., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, comprising 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2<0>C (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds, ‘Antisense Research and Applications’, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications. Modified nucleobases are described in US patent nos. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175, 273; 5, 367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,596,091; 5,614,617; 5,750,692, and 5,681,941, each of which is herein incorporated by reference.

In some embodiments, the ASOs are chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the ASO. Such moieties comprise but are not limited to, lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al, Ann. N. Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Mancharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-t oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937). See also U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552, 538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486, 603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762, 779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082, 830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5, 245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391, 723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5, 565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599, 928 and 5,688,941, each of which is herein incorporated by reference.

These moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention. Representative conjugate groups are disclosed in International Patent Application No. PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860, which are incorporated herein by reference. Conjugate moieties include, but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety. See, e.g., U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.

Because of the heterogeneity in human Alu sequences across the genome, the use of pools of ASOs that target multiple families may be desired. In some embodiments, ASOs comprising the following sequences are used: 5-GGCCGAGGCGGGCGG-3 (SEQ ID NO:71) and 5-TTTGGGAGGCCGAGG-3 (SEQ ID NO:72).

siRNA/shRNA

In some embodiments, the ASOs used in the present methods are interfering RNAs, including but not limited to a small interfering RNAs (“siRNAs”) or a small hairpin RNAs (“shRNAs”). Methods for constructing interfering RNAs are well known in the art. For example, the interfering RNA can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary (i.e., each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double stranded structure); the antisense strand comprises nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof (i.e., an undesired gene) and the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. Alternatively, interfering RNA is assembled from a single oligonucleotide, where the self-complementary sense and antisense regions are linked by means of nucleic acid based or non-nucleic acid-based linker(s). The interfering RNA can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The interfering can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating RNA interference.

In some embodiments, the interfering RNA coding region encodes a self-complementary RNA molecule having a sense region, an antisense region and a loop region. Such an RNA molecule when expressed desirably forms a “hairpin” structure, and is referred to herein as an “shRNA.” The loop region is generally between about 2 and about 10 nucleotides in length. In some embodiments, the loop region is from about 6 to about 9 nucleotides in length. In some embodiments, the sense region and the antisense region are between about 15 and about 20 nucleotides in length. Following post-transcriptional processing, the small hairpin RNA is converted into a siRNA by a cleavage event mediated by the enzyme Dicer, which is a member of the RNase III family. The siRNA is then capable of inhibiting the expression of a gene with which it shares homology. For details, see Brummelkamp et al., Science 296:550-553, (2002); Lee et al, Nature Biotechnol., 20, 500-505, (2002); Miyagishi and Taira, Nature Biotechnol 20:497-500, (2002); Paddison et al. Genes & Dev. 16:948-958, (2002); Paul, Nature Biotechnol, 20, 505-508, (2002); Sui, Proc. Natl. Acad. Sd. USA, 99(6), 5515-5520, (2002); Yu et al. Proc NatlAcadSci USA 99:6047-6052, (2002).

The target RNA cleavage reaction guided by siRNAs is highly sequence specific. In general, siRNA containing a nucleotide sequences identical to a portion of the target nucleic acid are preferred for inhibition. However, 100% sequence identity between the siRNA and the target gene is not required to practice the present invention. Thus the invention has the advantage of being able to tolerate sequence variations that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence. For example, siRNA sequences with insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for inhibition. Alternatively, siRNA sequences with nucleotide analog substitutions or insertions can be effective for inhibition. In general the siRNAs must retain specificity for their target, i.e., must not directly bind to, or directly significantly affect expression levels of, transcripts other than the intended target. Because of the heterogeneity in human Alu sequences across the genome, the use of pools of siRNAs that target multiple families may be desired.

Locked Nucleic Acids (LNAs)

In some embodiments, the modified ASOs used in the methods described herein comprise locked nucleic acid (LNA) molecules, e.g., including [alpha]-L-LNAs. LNAs comprise ribonucleic acid analogues wherein the ribose ring is “locked” by a methylene bridge between the 2′-oxgygen and the 4′-carbon—i.e., ASOs containing at least one LNA monomer, that is, one 2′-0,4′-C-methylene-fl-D-ribofuranosyl nucleotide. LNA bases form standard Watson-Crick base pairs but the locked configuration increases the rate and stability of the basepairing reaction (Jensen et al., Oligonucleotides, 14, 130-146 (2004)). LNAs also have increased affinity to base pair with RNA as compared to DNA and initiate cleavage by RNAse H. These properties render LNAs especially useful as probes for fluorescence in situ hybridization (FISH) and comparative genomic hybridization, as knockdown tools for miRNAs, and as antisense oligonucleotides to target mRNAs or other RNAs, e.g., RNAs as described herein. See, e.g., Kurreck et al., Nucleic Acids Res. 30(9): 1911-1918 (2002).

The LNA molecules can include molecules comprising 10-30, e.g., 12-24, e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand, wherein one of the strands is substantially identical, e.g., at least 80% (or more, e.g., 85%, 90%, 95%, or 100%) identical, e.g., having 3, 2, 1, or 0 mismatched nucleotide(s), to a target region in the RNA. The LNA molecules can be chemically synthesized using methods known in the art.

The LNA molecules can be designed using any method known in the art; a number of algorithms are known, and are commercially available (e.g., on the internet, for example at exiqon.com). See, e.g., You et al., Nuc. Acids. Res. 34:e60 (2006); McTigue et al., Biochemistry 43:5388-405 (2004); and Levin et al., Nuc. Acids. Res. 34:e142 (2006). For example, “gene walk” methods, similar to those used to design antisense oligos, can be used to optimize the inhibitory activity of the LNA; for example, a series of ASOs of 10-30 nucleotides spanning the length of a target RNA can be prepared, followed by testing for activity. Optionally, gaps, e.g., of 5-10 nucleotides or more, can be left between the LNAs to reduce the number of ASOs synthesized and tested. GC content is preferably between about 30-60%. General guidelines for designing LNAs are known in the art; for example, LNA sequences will bind very tightly to other LNA sequences, so it is preferable to avoid significant complementarity within an LNA. Contiguous runs of more than four LNA residues, should be avoided where possible (for example, it may not be possible with very short (e.g., about 9-10 nt) ASOs). In some embodiments, the LNAs are xylo-LNAs. For additional information regarding LNAs see U.S. Pat. Nos. 6,268,490; 6,734,291; 6,770,748; 6,794,499; 7,034,133; 7,053,207; 7,060,809; 7,084,125; and 7,572,582; and U.S. Pre-Grant Pub. Nos. 20100267018; 20100261175; and 20100035968; Koshkin et al. Tetrahedron 54, 3607-3630 (1998); Obika et al. Tetrahedron Lett. 39, 5401-5404 (1998); Jepsen et al., Oligonucleotides 14:130-146 (2004); Kauppinen et al., Drug Disc. Today 2(3):287-290 (2005); and Ponting et al., Cell 136(4):629-641 (2009), and references cited therein.

Because of the heterogeneity in human Alu sequences across the genome, the use of pools of LNAs that target multiple families may be desired.

Making and Using ASOs

Nucleic acid sequences used to practice this invention can be made using methods known in the art, e.g., synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett. 22:1859; U.S. Pat. No. 4,458,066.

Techniques for the manipulation of nucleic acids used to practice this invention, such as, e.g., subcloning, labeling probes (e.g., random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature, see, e.g., Sambrook et al., Molecular Cloning; A Laboratory Manual 3d ed. (2001); Current Protocols in Molecular Biology, Ausubel et al., eds. (John Wiley & Sons, Inc., New York 2010); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); Laboratory Techniques In Biochemistry And Molecular Biology: Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).

Alu/B2 Nucleic Acids

The methods described herein can also include the use of Alu or B2 nucleic acids to induce cell death in a cell, e.g., for the treatment of disorders associated with abnormal apoptotic or differentiative processes. The Alu or B2 nucleic acids can be, e.g., Alu or B2 RNA comprising a full length Alu or B2 sequence, or a fragment thereof that induces cell death. Methods for identifying fragments that induce cell death are known in the art and described herein, see, e.g., Example 3 herein. The methods can include incubating a sample of test cells, e.g., cancer cells, in the presence of a candidate fragment and a control fragment (e.g., of the same length and modifications but having a scrambled sequence), and selecting those fragments that induce cell death under conditions in which the control fragment does not induce cell death.

The Alu or B2 nucleic acids can be administered to the cells as RNA, e.g., naked RNA or RNA encapsulated in a carrier, e.g., a liposomal carrier. Alternatively, an expression construct encoding the Alu or B2 nucleic acid or fragment thereof can be administered.

Expression Constructs

Expression constructs encoding an Alu or B2 nucleic acid or fragment thereof can be administered in any effective carrier, e.g., any formulation or composition capable of effectively delivering the component gene to cells in vivo. Approaches include insertion of the gene in viral vectors, including recombinant retroviruses, adenovirus, adeno-associated virus, lentivirus, and herpes simplex virus-1, or recombinant bacterial or eukaryotic plasmids. Viral vectors transfect cells directly; plasmid DNA can be delivered naked or with the help of, for example, cationic liposomes (lipofectamine) or derivatized (e.g., antibody conjugated), polylysine conjugates, gramacidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or CaPO₄ precipitation carried out in vivo. A preferred approach for in vivo introduction of nucleic acid into a cell is by use of a viral vector containing nucleic acid, e.g., a cDNA. Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid. Additionally, molecules encoded within the viral vector, e.g., by a cDNA contained in the viral vector, are expressed efficiently in cells that have taken up viral vector nucleic acid.

Retrovirus vectors and adeno-associated virus vectors can be used as a recombinant gene delivery system for the transfer of exogenous genes in vivo, particularly into humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host. The development of specialized cell lines (termed “packaging cells”) which produce only replication-defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses are characterized for use in gene transfer for gene therapy purposes (for a review see Miller, Blood 76:271 (1990)). A replication defective retrovirus can be packaged into virions, which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Ausubel, et al., eds., Current Protocols in Molecular Biology, Greene Publishing Associates, (1989), Sections 9.10-9.14, and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are known to those skilled in the art. Examples of suitable packaging virus lines for preparing both ecotropic and amphotropic retroviral systems include ΨCrip, ΨCre, Ψ2 and ΨAm. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro and/or in vivo (see for example Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol. 150:4104-4115; U.S. Pat. Nos. 4,868,116; 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573).

Another viral gene delivery system useful in the present methods utilizes adenovirus-derived vectors. The genome of an adenovirus can be manipulated, such that it encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See, for example, Berkner et al., BioTechniques 6:616 (1988); Rosenfeld et al., Science 252:431-434 (1991); and Rosenfeld et al., Cell 68:143-155 (1992). Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 d1324 or other strains of adenovirus (e.g., Ad2, Ad3, or Ad7 etc.) are known to those skilled in the art. Recombinant adenoviruses can be advantageous in certain circumstances, in that they are not capable of infecting non-dividing cells and can be used to infect a wide variety of cell types, including epithelial cells (Rosenfeld et al., (1992) supra). Furthermore, the virus particle is relatively stable and amenable to purification and concentration, and as above, can be modified so as to affect the spectrum of infectivity. Additionally, introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situ, where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA). Moreover, the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al., supra; Haj-Ahmand and Graham, J. Virol. 57:267 (1986).

Yet another viral vector system useful for delivery of nucleic acids is the adeno-associated virus (AAV). Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle. (For a review see Muzyczka et al., Curr. Topics in Micro. and Immunol. 158:97-129 (1992). It is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (see for example Flotte et al., Am. J. Respir. Cell. Mol. Biol. 7:349-356 (1992); Samulski et al., J. Virol. 63:3822-3828 (1989); and McLaughlin et al., J. Virol. 62:1963-1973 (1989). Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate. Space for exogenous DNA is limited to about 4.5 kb. An AAV vector such as that described in Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985) can be used to introduce DNA into cells. A variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al., Proc. Natl. Acad. Sci. USA 81:6466-6470 (1984); Tratschin et al., Mol. Cell. Biol. 4:2072-2081 (1985); Wondisford et al., Mol. Endocrinol. 2:32-39 (1988); Tratschin et al., J. Virol. 51:611-619 (1984); and Flotte et al., J. Biol. Chem. 268:3781-3790 (1993).

In some embodiments, Alu or B2 nucleic acid or fragments thereof, or nucleic acids encoding an Alu or B2 nucleic acid or fragments thereof, are entrapped in liposomes bearing positive charges on their surface (e.g., lipofectins), which can be tagged with antibodies against cell surface antigens of the target cancer cells.

In clinical settings, the nucleic acids can be introduced into a subject by any of a number of methods, each of which is familiar in the art. For instance, a pharmaceutical preparation can be introduced systemically, e.g., by intravenous injection, and specific transduction of the protein in the target cells will occur predominantly from specificity of transfection, provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the receptor gene, or a combination thereof. In other embodiments, initial delivery of the nucleic acids is more limited, with introduction into the subject being quite localized. For example, the nucleic acids can be introduced by catheter (see U.S. Pat. No. 5,328,470) or by stereotactic injection (e.g., Chen et al., PNAS USA 91: 3054-3057 (1994)). In some embodiments, the nucleic acids are administered during or after surgical resection of a tumor; in some embodiments, a controlled-release hydrogel comprising the nucleic acids is administered at the conclusion of resection before closure to provide a steady dose of the nucleic acids over time.

A pharmaceutical preparation of the nucleic acids can consist essentially of the gene delivery system (e.g., viral vector(s)) in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is embedded. Alternatively, where the complete gene delivery system can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can comprise one or more cells, which produce the gene delivery system.

Treating Cellular Differentiative Disorders

As noted above, the methods described herein can also include the use of Alu or B2 nucleic acids or fragments thereof to induce cell death in a cell, e.g., for the treatment of disorders associated with abnormal apoptotic or differentiative processes, e.g., cellular proliferative disorders or cellular differentiative disorders, e.g., cancer, e.g., by producing an active or passive immunity. Examples of cellular proliferative and/or differentiative disorders include cancer, e.g., carcinoma, sarcoma, metastatic disorders or hematopoietic neoplastic disorders, e.g., leukemias. A metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of prostate, colon, lung, breast and liver origin.

As used herein, the terms “cancer”, “hyperproliferative” and “neoplastic” refer to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. Hyperproliferative and neoplastic disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. “Pathologic hyperproliferative” cells occur in disease states characterized by malignant tumor growth. Examples of non-pathologic hyperproliferative cells include proliferation of cells associated with wound repair. The terms “cancer” or “neoplasms” include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.

The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. In some embodiments, the disease is renal carcinoma or melanoma. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.

The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.

Additional examples of proliferative disorders include hematopoietic neoplastic disorders. As used herein, the term “hematopoietic neoplastic disorders” includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin, e.g., arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof. Preferably, the diseases arise from poorly differentiated acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia. Additional exemplary myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit Rev. in Oncol./Hemotol. 11:267-97); lymphoid malignancies include, but are not limited to acute lymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM). Additional forms of malignant lymphomas include, but are not limited to non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Sternberg disease.

Other examples of proliferative and/or differentiative disorders include skin disorders. The skin disorder may involve the aberrant activity of a cell or a group of cells or layers in the dermal, epidermal, or hypodermal layer, or an abnormality in the dermal-epidermal junction. For example, the skin disorder may involve aberrant activity of keratinocytes (e.g., hyperproliferative basal and immediately suprabasal keratinocytes), melanocytes, Langerhans cells, Merkel cells, immune cell, and other cells found in one or more of the epidermal layers, e.g., the stratum basale (stratum germinativum), stratum spinosum, stratum granulosum, stratum lucidum or stratum corneum. In other embodiments, the disorder may involve aberrant activity of a dermal cell, e.g., a dermal endothelial, fibroblast, immune cell (e.g., mast cell or macrophage) found in a dermal layer, e.g., the papillary layer or the reticular layer. Examples of skin disorders include psoriasis, psoriatic arthritis, dermatitis (eczema), e.g., exfoliative dermatitis or atopic dermatitis, pityriasis rubra pilaris, pityriasis rosacea, parapsoriasis, pityriasis lichenoiders, lichen planus, lichen nitidus, ichthyosiform dermatosis, keratodermas, dermatosis, alopecia areata, pyoderma gangrenosum, vitiligo, pemphigoid (e.g., ocular cicatricial pemphigoid or bullous pemphigoid), urticaria, prokeratosis, rheumatoid arthritis that involves hyperproliferation and inflammation of epithelial-related cells lining the joint capsule; dermatitises such as seborrheic dermatitis and solar dermatitis; keratoses such as seborrheic keratosis, senile keratosis, actinic keratosis. photo-induced keratosis, and keratosis follicularis; acne vulgaris; keloids and prophylaxis against keloid formation; nevi; warts including verruca, condyloma or condyloma acuminatum, and human papilloma viral (HPV) infections such as venereal warts; leukoplakia; lichen planus; and keratitis. The skin disorder can be dermatitis, e.g., atopic dermatitis or allergic dermatitis, or psoriasis.

In some embodiments, the disorder is psoriasis. The term “psoriasis” is intended to have its medical meaning, namely, a disease which afflicts primarily the skin and produces raised, thickened, scaling, nonscarring lesions. The lesions are usually sharply demarcated erythematous papules covered with overlapping shiny scales. The scales are typically silvery or slightly opalescent. Involvement of the nails frequently occurs resulting in pitting, separation of the nail, thickening and discoloration. Psoriasis is sometimes associated with arthritis, and it may be crippling. Hyperproliferation of keratinocytes is a key feature of psoriatic epidermal hyperplasia along with epidermal inflammation and reduced differentiation of keratinocytes. Multiple mechanisms have been invoked to explain the keratinocyte hyperproliferation that characterizes psoriasis. Disordered cellular immunity has also been implicated in the pathogenesis of psoriasis. Examples of psoriatic disorders include chronic stationary psoriasis, psoriasis vulgaris, eruptive (gluttate) psoriasis, psoriatic erythroderma, generalized pustular psoriasis (Von Zumbusch), annular pustular psoriasis, and localized pustular psoriasis.

Pharmaceutical Compositions

The methods described herein can include the administration of pharmaceutical compositions and formulations comprising an Alu or B2 RNA, a DNA encoding an Alu or B2 RNA, or an ASO that targets Alu or B2 RNA.

In some embodiments, the compositions are formulated with a pharmaceutically acceptable carrier. The pharmaceutical compositions and formulations can be administered parenterally, topically, orally or by local administration, such as by aerosol or transdermally. The pharmaceutical compositions can be formulated in any way and can be administered in a variety of unit dosage forms depending upon the condition or disease and the degree of illness, the general medical condition of each patient, the resulting preferred method of administration and the like. Details on techniques for formulation and administration of pharmaceuticals are well described in the scientific and patent literature, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005.

The ASOs can be administered alone or as a component of a pharmaceutical formulation (composition). The compounds may be formulated for administration, in any convenient way for use in human or veterinary medicine. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Formulations of the compositions of the invention include those suitable for intradermal, inhalation, oral/nasal, topical, parenteral, rectal, and/or intravaginal administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient (e.g., nucleic acid sequences of this invention) which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration, e.g., intradermal or inhalation. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect, e.g., an antigen specific T cell or humoral response.

Pharmaceutical formulations can be prepared according to any method known to the art for the manufacture of pharmaceuticals. Such drugs can contain sweetening agents, flavoring agents, coloring agents and preserving agents. A formulation can be admixtured with nontoxic pharmaceutically acceptable excipients which are suitable for manufacture. Formulations may comprise one or more diluents, emulsifiers, preservatives, buffers, excipients, etc. and may be provided in such forms as liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled release formulations, tablets, pills, gels, on patches, in implants, etc.

Pharmaceutical formulations for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in appropriate and suitable dosages. Such carriers enable the pharmaceuticals to be formulated in unit dosage forms as tablets, pills, powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient. Pharmaceutical preparations for oral use can be formulated as a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable additional compounds, if desired, to obtain tablets or dragee cores. Suitable solid excipients are carbohydrate or protein fillers include, e.g., sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxy-methylcellulose; and gums including arabic and tragacanth; and proteins, e.g., gelatin and collagen. Disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate. Push-fit capsules can contain active agents mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active agents can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.

Aqueous suspensions can contain an active agent (e.g., nucleic acid sequences of the invention) in admixture with excipients suitable for the manufacture of aqueous suspensions, e.g., for aqueous intradermal injections. Such excipients include a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethylene oxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensation product of ethylene oxide with a partial ester derived from fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate). The aqueous suspension can also contain one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose, aspartame or saccharin. Formulations can be adjusted for osmolarity.

In some embodiments, oil-based pharmaceuticals are used for administration of nucleic acid sequences of the invention. Oil-based suspensions can be formulated by suspending an active agent in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin; or a mixture of these. See e.g., U.S. Pat. No. 5,716,928 describing using essential oils or essential oil components for increasing bioavailability and reducing inter- and intra-individual variability of orally administered hydrophobic pharmaceutical compounds (see also U.S. Pat. No. 5,858,401). The oil suspensions can contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents can be added to provide a palatable oral preparation, such as glycerol, sorbitol or sucrose. These formulations can be preserved by the addition of an antioxidant such as ascorbic acid. As an example of an injectable oil vehicle, see Minto (1997) J. Pharmacol. Exp. Ther. 281:93-102.

Pharmaceutical formulations can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil, described above, or a mixture of these. Suitable emulsifying agents include naturally-occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan mono-oleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. The emulsion can also contain sweetening agents and flavoring agents, as in the formulation of syrups and elixirs. Such formulations can also contain a demulcent, a preservative, or a coloring agent. In alternative embodiments, these injectable oil-in-water emulsions of the invention comprise a paraffin oil, a sorbitan monooleate, an ethoxylated sorbitan monooleate and/or an ethoxylated sorbitan trioleate.

The pharmaceutical compounds can also be administered by in intranasal, intraocular and intravaginal routes including suppositories, insufflation, powders and aerosol formulations (for examples of steroid inhalants, see e.g., Rohatagi (1995) J. Clin. Pharmacol. 35:1187-1193; Tjwa (1995) Ann. Allergy Asthma Immunol. 75:107-111). Suppositories formulations can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at body temperatures and will therefore melt in the body to release the drug. Such materials are cocoa butter and polyethylene glycols.

In some embodiments, the pharmaceutical compounds can be delivered transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols. In some embodiments, the pharmaceutical compounds can also be delivered as microspheres for slow release in the body. For example, microspheres can be administered via intradermal injection of drug which slowly release subcutaneously; see Rao (1995) J. Biomater Sci. Polym. Ed. 7:623-645; as biodegradable and injectable gel formulations, see, e.g., Gao (1995) Pharm. Res. 12:857-863 (1995); or, as microspheres for oral administration, see, e.g., Eyles (1997) J. Pharm. Pharmacol. 49:669-674.

In some embodiments, the pharmaceutical compounds can be parenterally administered, such as by intravenous (IV) administration or administration into a body cavity or lumen of an organ. These formulations can comprise a solution of active agent dissolved in a pharmaceutically acceptable carrier. Acceptable vehicles and solvents that can be employed are water and Ringer's solution, an isotonic sodium chloride. In addition, sterile fixed oils can be employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can likewise be used in the preparation of injectables. These solutions are sterile and generally free of undesirable matter. These formulations may be sterilized by conventional, well known sterilization techniques. The formulations may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of active agent in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the patient's needs. For IV administration, the formulation can be a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated using those suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a suspension in a nontoxic parenterally-acceptable diluent or solvent, such as a solution of 1,3-butanediol. The administration can be by bolus or continuous infusion (e.g., substantially uninterrupted introduction into a blood vessel for a specified period of time).

In some embodiments, the pharmaceutical compounds and formulations can be lyophilized. Stable lyophilized formulations comprising an ASO can be made by lyophilizing a solution comprising a pharmaceutical of the invention and a bulking agent, e.g., mannitol, trehalose, raffinose, and sucrose or mixtures thereof. A process for preparing a stable lyophilized formulation can include lyophilizing a solution about 2.5 mg/mL protein, about 15 mg/mL sucrose, about 19 mg/mL NaCl, and a sodium citrate buffer having a pH greater than 5.5 but less than 6.5. See, e.g., U.S. 20040028670.

The compositions and formulations can be delivered by the use of liposomes. By using liposomes, particularly where the liposome surface carries ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the active agent into target cells in vivo. See, e.g., U.S. Pat. Nos. 6,063,400; 6,007,839; Al-Muhammed (1996) J. Microencapsul. 13:293-306; Chonn (1995) Curr. Opin. Biotechnol. 6:698-708; Ostro (1989) Am. J. Hosp. Pharm. 46:1576-1587. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes that are believed to interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH-sensitive or negatively-charged are believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells.

Liposomes can also include “sterically stabilized” liposomes, i.e., liposomes comprising one or more specialized lipids. When incorporated into liposomes, these specialized lipids result in liposomes with enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Liposomes and their uses are further described in U.S. Pat. No. 6,287,860.

The formulations of the invention can be administered for prophylactic and/or therapeutic treatments. In some embodiments, for therapeutic applications, compositions are administered to a subject who is need of reduced triglyceride levels, or who is at risk of or has a disorder described herein, in an amount sufficient to cure, alleviate or partially arrest the clinical manifestations of the disorder or its complications; this can be called a therapeutically effective amount. For example, in some embodiments, pharmaceutical compositions of the invention are administered in an amount sufficient to decrease serum levels of triglycerides in the subject. The amount of pharmaceutical composition adequate to accomplish this is a therapeutically effective dose. The dosage schedule and amounts effective for this use, i.e., the dosing regimen, will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient's physical status, age and the like. In calculating the dosage regimen for a patient, the mode of administration also is taken into consideration.

The dosage regimen also takes into consideration pharmacokinetics parameters well known in the art, i.e., the active agents' rate of absorption, bioavailability, metabolism, clearance, and the like (see, e.g., Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:611-617; Groning (1996) Pharmazie 51:337-341; Fotherby (1996) Contraception 54:59-69; Johnson (1995) J. Pharm. Sci. 84:1144-1146; Rohatagi (1995) Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24:103-108; Remington: The Science and Practice of Pharmacy, 21st ed., 2005). The state of the art allows the clinician to determine the dosage regimen for each individual patient, active agent and disease or condition treated. Guidelines provided for similar compositions used as pharmaceuticals can be used as guidance to determine the dosage regiment, i.e., dose schedule and dosage levels, administered practicing the methods of the invention are correct and appropriate.

Single or multiple administrations of formulations can be given depending on for example: the dosage and frequency as required and tolerated by the patient, the degree and amount of therapeutic effect generated after each administration (e.g., effect on tumor size or growth), and the like. The formulations should provide a sufficient quantity of active agent to effectively treat, prevent or ameliorate conditions, diseases or symptoms.

In alternative embodiments, pharmaceutical formulations for oral administration are in a daily amount of between about 1 to 100 or more mg per kilogram of body weight per day. Lower dosages can be used, in contrast to administration orally, into the blood stream, into a body cavity or into a lumen of an organ. Substantially higher dosages can be used in topical or oral administration or administering by powders, spray or inhalation. Actual methods for preparing parenterally or non-parenterally administrable formulations will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington: The Science and Practice of Pharmacy, 21st ed., 2005.

In some embodiments, the methods described herein can include co-administration with other drugs or pharmaceuticals, e.g., compositions for providing cholesterol homeostasis. For example, the ASOs can be co-administered with drugs for treating or reducing risk of a disorder described herein.

EXAMPLES

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Experimental Procedures

The following materials and methods were used in the Examples below.

Cell culture and transfections. NIH/3T3 cells were cultured in DMEM+Glutamax (Life Technologies) supplemented with 10% fetal bovine serum and 1% Penicillin/Streptomycin. Before heat shock stimulus cells were trypsinized and resuspended in 5 ml complete medium in a 15 ml falcon tube. Subsequently, cells were either placed in 37° C. (control cells, pre-H/S condition) or in 45° C. (treated cells, post-H/S condition) for 15 min. Time points mentioned throughout this work have as a starting point the moment of the start of the heat shock stimulus. After the end of this 15 minute period, cells were centrifuged shortly (2 min) and cell pellets were directly resuspended into Trizol (Thermofischer) for the RNA-seq analysis or fixated with 1% formaldehyde for the ChIP-seq and CHART-seq analysis. For LNA transfections against B2 RNA we used the HiPerfect transfection reagent (Qiagen) and the sequence of the LNAs used were as follows: LNA11: 5′-GTTACGGATGGTTGTG-3′ and LNA12: 5′-TGTAGCTGTCTTCAG-3′. The scramble LNA sequence was 5′-CACGTCTATACACCAC-3′. In detail, the LNAs were diluted to 100 uM and incubated with 1.35 ul of the transfection reagent in a final volume of 10 ul for 15-20 min at room temperature (RT). Subsequently the transfection mix was transferred to 2 ml of recently trypsinized cells in full culture medium containing 5×10⁵ cells (final LNA concentration 500 nM). A fluorophore conjugated LNA was also transfected to test transfection efficiency. Subsequently cells were plated and incubated at 37° C. for 24 hours before testing. In the meanwhile, after 1 h from plating, a subset of cells was subjected to FACS analysis and transfection rate was estimated to 90% of live cells. For LNA transfections against Ezh2 we used the following LNA ASO sequence: 5′-TTCTTCTTCTGTGCAG-3′. Transfections were performed with HiPerfect as mentioned above but for a final LNA concentration of 25 nM. For RNA transfections of the B2 RNA and its fragments we used the TransMessenger Transfection Reagent (Qiagen). In brief, 16 pmol of RNA in Buffer EC was incubated for 5 min at RT with 2 ul enhancer, and subsequently 8 ul transfection reagent was added to a total reaction of 100 ul and incubated for 10 min at RT before addition to recently trypsinized cells in culture medium without serum. 2,6×10⁴ transfected cells were plated and incubated at 37° C. for 30 min before adding an equal volume of complete medium (with serum). After 2 hours, a subset of these cells were washed with PBS twice and RNA was extracted using Trizol and analyzed with qPCR against B2 RNA to confirm B2 overexpression. After 6 hours from plating the medium was changed to complete medium and cells were counted during the subsequent days using a Nexcelom Cellometer.

RNA in vitro transcription and RNA-protein incubations. RNAs were transcribed in vitro and Ezh2, Eed and GST proteins were purified as described previously (32) with the following modifications: For RNA in vitro transcription we used the AmpliScribe T7 High Yield Transcription Kit (Epicentre) applying a 3 h incubation at 42° C. and using a template resulting to the following B2 RNA sequence: 5′-GGGGCTGGTGAGATGGCTCAGTGGGTAAGAGCACCCGACTGCTCTTCCGA AGGTCCGGAGTTCAAATCCCAGCAACCACATGGTGGCTCACAACCATCCG TAACGAGATCTGACTCCCTCTTCTGGAGTGTCTGAAGACAGCTACAGTGT ACTTACATATAATAAATAAATAAATCTTTAAAAAAAAA-3′.

For smaller B2 RNA fragments the respective templates were constructed based on the above sequence and the nt numbering mentioned in the text. In detail, domain I RNA was from +lnt to +72 nt, domain RNA from +lnt to +105 nt, and domain III from +99 to +140 nt. The quality of the transcribed RNA was tested running a 6% UREA PAGE gel as well as through small RNA-seq library construction and next generation sequencing (see below). RNAs were purified using the ZymoResearch RNA clean kit. Incubations, unless mentioned differently in the text were performed with 200 nM in-vitro-transcribed B2 RNA folded with 300 mM NaCl and supplemented with TAP buffer (final reaction concentrations: 5 nM Tris pH 7.9, 0.5 mM MgCl2, 0.02 mM EDTA, 0.01% NP40, 1% glycerol, 0.2 mM DTT). For RNA folding the RNA was incubated for 1 min at 50° C. and cooled down with a rate of 1° C./10 sec. Cleavage time-courses were quantified using ImageJ (NIH). The fraction of full-Length B2 RNA present at each time point was measured and this data was fit using Kaleidagraph (Synergy) using the differential form of the rate equation for an irreversible, first-order reaction.

Double-Filter Binding Assays. Binding reactions were assembled with 1 μl of 1,000 cpm/μl (0.1 nM final concentration) folded RNA and purified protein at the shown concentrations in binding buffer (50 mM Tris-HCl [pH 8.0], 100 mM NaCl, 5 mM MgCl2, 10 μg/ml BSA, 0.05% NP40, 1 mM DTT, 20 U RNaseOUT [Invitrogen], and 5% glycerol) in 30 μl. A total of 50 ng/μl yeast tRNA (Ambion catalog number AM7119) was used as a nonspecific competitor. After 30 min at 30° C., the reactions were filtered through nitrocellulose (PROTRAN, Schleicher & Schuell) and Hybond-N+(GE Healthcare) membranes using a Minifold I system (Whatman), washed with 600 μl washing buffer (50 mM Tris-HCl [pH 8.0], 100 mM NaCl, 1.5 mM MgCl2, 0.05% NP40, 1 mM DTT), dried, exposed to a phosphor screen, and scanned after 2 hr in a Typhoon Trio (GE Healthcare Life Sciences). Data were quantified by Quantity One and normalized as previously described (Cifuentes-Rojas et al., 2014). Equilibrium dissociation constants, Kd, were obtained by fitting the binding data to a one-site binding model by nonlinear regression using Graphpad Prism.

CHART and ChIP analyses. At least two biological replicates were analyzed for CHART and ChIP experiments. The B2 CHART was modified from the original CHART protocols (33). In detail, 12 millions cells were crosslinked with 1% formaldehyde for 10 min at room temperature. Crosslinking was then quenched with 0.125 M glycine for 5 min and washed with PBS 3 times. Snap freezing cells could be stored at −80° C. Crosslinked cells were re-suspended in 2 ml of sucrose buffer (0.3 M sucrose, 1% Triton-X-100, 10 mM HEPES pH 7.5, 100 mM KOAc, 0.1 mM EGTA), dounced 20 times with a tight pestle, and kept on ice for 10 min. The following steps were using polystyrene tubes, glass pipettes, and DNA LoBind microtubes (Eppendorf) to avoid cell clumps sticking onto the walls of tubes or pipettes. Nuclei were collected by centrifugation at 1,500 g for 10 min on top of a cushion of 5 ml glycerol buffer (25% glycerol, 10 mM HEPES pH7.5, 1 mM EDTA, 0.1 mM EGTA, 100 mM KOAc). Nuclei were further crosslinked with 3% formaldehyde for 30 min at room temperature. After washing three times with ice-cold PBS, nuclei were extracted once with 50 mM HEPES pH7.5, 250 mM NaCl, 0.1 mM EGTA, 0.5% N-lauroylsarcosine, 0.1% sodium deoxycholate, 5 mM DTT, 100 U/ml SUPERasIN (Invitrogen) for 10 min on ice, and centrifuged at 400 g for 5 min at 4° C. Nuclei were resuspended in 1.2 ml of sonication buffer (50 mM HEPES pH 7.5, 75 mM NaCl, 0.1 mM EGTA, 0.5% N-lauroylsarcosine, 0.1% sodium deoxycholate, 5 mM DTT, 10 U/ml SUPERasIN, and sonicated in microtubes using Covaris E220 sonicator at 10% duty cycle, 200 bursts per cycle, 105 peak intensity power for 5 min. The major size of chromatin fragments was around 3-4 kb. Fragmented chromatin was subjected to hybridization immediately. Hybridization, washing and elution were performed as follows. In brief, beads were blocked with 500 ng/ul yeast total RNA, and 1 mg/ml BSA for 1 hr at 37° C., and respuspended in 1× hybridization buffer. 360 μl of 2× hybridization buffer (750 mM NaCl, 1% SDS, 50 mM Tris pH 7.0, 1 mM EDTA, 15% Formamide, 1 mM DTT, PMSF, protease inhibitor, and 100 U/ml Superase-in) was added into 180 μl lysates, and then this 1× hybridization lysate was precleaned by 60 μl of blocked beads at room temperature for 1 hr. After removal of the beads, B2 probes (labeled with 3′ biotin-TEG, 18 pmol) for B2 RNA were added into the 1× hybridization lysate and incubate at room temperature for overnight. Given the variability of the different B2 repeats, we used a pool of probes that correspond to the majority of the sequence variations within the target region presented at FIG. 4a . As control we used also a negative probe that does not show any sequence similarity to the used probes with he following sequence: 5-GCACGTCTATACACCACT-3′. 120 ul of blocked beads were added into lysates and incubated at RT for two hours. Beads:biotin-probes:RNA:chromatin adducts were captured by magnets, washed once with 1× hybridization buffer at 37° C. for 30 min, washed four times at 37° C. for 5 min with SDS wash buffer (2×SSC, 1% SDS, 1 mM DTT, 1 mM PMSF), and then washed once for 5 min at room temperature with 0.1% NP40 buffer (150 mM NaCl, 50 mM Tris pH8.0, 3 mM MgCl2, 10 mM DTT, 0.1% NP40). DNA was then eluted in 100 μl twice for 20 min in 100 μl of 0.1% NP40 buffer with 200 U/ml RNase H (NEB) at room temperature and purified further using phenol-chloroform extraction. Before ChIP analysis, 3 millions cells were crosslinked as above and sheared chromatin was prepared using the ChIP-IT Express kit (Active motif) in a 135 ul volume using the following conditions in a Covaris E220 sonicator: 2% duty cycle, 200 bursts per cycle, 105 peak intensity power for 5 min. Chromatin immunoprecipitations were performed in 100 ul reaction volumes using the same kit as with chromatin shearing and the following antibodies for 14 h incubation times: Ezh2 (D2C9, 5246S Cell signaling technology), H3K27me3 (39155, active motif), RNA pol II phospho S2 (from the ab103968 panel, abcam), RNA pol II phospho S5 (from the ab103968 panel, abcam), Hsf1P (ADI-SPA-901-D, Enzo life sciences). Eluted DNA was further purified with phenol-chloroform.

Library construction for RNA sequencing. RNA used for short RNA-seq and RNA-seq libraries was prepared as follows: Total RNA from cells was extracted using Trizol and 4 ug of total RNA was subjected to ribosomal RNA depletion using the ribominus V2 kit (Life technologies). Incubation of the RNA with the probe was done for 40 min instead of 20 min. RNA depleted RNA was separated into two fractions of short (<200) and longer RNAs using the mirVana separation kit (Life technologies) with the following modifications: After addition of the lysis/binding buffer and the miRNA homogenate additive solution, 100% EtOH at 1/3 of the volume was added and the mix was passed through the filter to bind long RNAs. The flow through was collected and 100% EtOH at 2/3 of the flow through volume was added and passed through a new filter column to bind short RNAs. Elution of the long and short RNAs from each column respectively was done per manufacturer instructions. Eluted RNAs were concentrated in both cases using the RNeasy MinElute Spin Columns (Qiagen) and tested for its size and quality using an Agilent Bioanalyzer RNA kit. For short RNA library construction, ribo-depleted short RNAs were subjected to PNK phosphorylation for 1 h at 37 C. Subsequently we used the NEBnext small RNA library construction kit (NEB) with the following modifications: Incubation of the 3′adaptor was performed for 2 h, and the libraries at the end were not subjected to double size selection with the Ampure beads but with 1,2× size selection. For sequencing of the in vitro B2 fragments no ribosomal depletion was applied For the longer RNAs we used the NEBNext Ultra directional RNA library kit (NEB) with an RNA fragmentation of 10 min at 95 C and with the following modifications: First strand synthesis at 42 C was done for 50 min and the End Prep of cDNA library was followed by an Ampure Beads selection of 1,8× and ligation of the adapters using the 5× quick ligation buffer and Quick T4 DNA ligase (NEB) for 30 min. Incubation with the USER enzyme was done before the PCR amplification for 30 min, followed by a double size selection of 0.5×-1×, while the final library was size selected using Ampure beads at a 1× sample-beads ratio. Libraries were evaluated using the Bioanalyser high sensitivity DNA kit (Agilent) and quantitated using the qPCR KAPPA kit (Kappa).

Library construction for ChIP and CHART sequencing. Purified DNA was subjected to further fragmentation in a Covaris E220 sonicator using 10% duty cycle, 200 bursts per cycle, 175 peak intensity power for 5 min in 125 ul. Subsequently, we used the NEBNext ChIP-seq library Prep Master MIX set (NEB) with the following modifications: For ChIP-seq the EndRepair of ChIP DNA was performed only for 15 min in a 10.5 ul total volume (using lul buffer and 0.5 ul enzyme) followed by no cleanup but dA-Tailing in a reaction scaled to 100 ul for 15 min. Subsequently we performed double size selection 0.2×-2.5× before adaptor (0.3 uM) ligation for 30 min and USER enzyme incubation for another 30 min. Ligation reaction was cleaned usingl.4 sample-bead ratio and the final library was size selected and clean with Ampure beads twice using 1× and 0.5×-0.9× ratios. In addition, the PCR reaction had an extension time of lmin and 30 sec. For CHART-seq the end repair was scaled to 150 ul, while the dA-tailing was performed at 25.5 ul total volume. After adaptor ligation it was size selected with 0.6×-1.2× bead-sample ratio, while after the PCR it was cleaned twice with 1× and 0.9× Ampure beads and quantified using the qPCR KAPPA kit.

Bioinformatics analysis. Raw RIP-seq, CHART-seq and ChIP-seq reads and the respective sequenced input reads were mapped using bwa.0.5.5 (Li and Durbin, 2010) (default parameters). Using in home scripts and bedtools (Quinlan and Hall, 2010) the resulting sam files were converted to bed files and enriched genomics regions against the input were filtered using SICER (Xu et al., 2014) with a window and gap parameter of 300 and an FDR 0.05. Subsequently, CHART-seq reads of the B2 probe were filtered further based on distribution of reads captured by the negative CHART probe. Metagene profiles were constructed using the Babraham NGS analysis suite Seqmonk (www.bioinformatics.babraham.ac.uk/projects_seqmonk/) employing normalized cumulative distributions filtered in case of CHART-reads against the positions of B2 elements (3 KB radius). Normalization was performed based on the total number of mapped reads. Seqmonk genome browser was used for visualization using RefSeq and RepeatMasker annotations for mRNAs and B2 SINE elements, respectively. Peak annotation was done using Galaxy (Afgan et al., 2016) and PAVIS (Huang et al., 2013).

Short RNA reads were trimmed from adapters in both ends using cutadapt (doi.org/10.14806/ej.17.1.200) for the following adapter sequences: AAGATCGGAAGAGCACACGTCT. Subsequently reads were mapped using bwa and converted to bed files with bedtools. Then, using in home transcripts 5′ ends coordinates of the reads were extracted and plotted against a metagene representing the absolute distance between start of B2 repeats and downstream sequences. Reads distributions and alignments were performed using seqmonk. Raw RNA-seq reads were trimmed using cutadapt for the following adapter sequences: AAGATCGGAAGAGCACACGTCT and AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT for read 1 and read 2, respectively. Subsequently they were mapped against mm9 reference transcriptome using tophat (Trapnell et al., 2009) with the following parameters: bowtie1-r-100-N 20—read-gap-length 10—segment-mismatches 3—read-edit-dist 20. Subsequently, differential expression was performed using Seqmonk's intensify difference function for a p value less than 0.05. Metagene profiles were plotted with Seqmonk using the relative read density function. Transcriptional start site was defined using the TSS Eponine track from Seqmonk (Down and Hubbard, 2002). Aread coverage was calculated (CoveragePostH/S−CoveragePreH/S)/((CoveragePostH/S+CoveragePreH/S)/2). Genome browser screenshots were derived using the IGV viewer (Robinson et al., 2011). For the statistical analysis of the read distributions we applied the Kolmogorov-Smirnov test, using Prism6 (Graphpad). Datasets for short-RNA-seq, RNA-seq, ChIP-seq and CHART-seq have been deposited in GEO (GSE82255).

Example 1. B2 RNA Associates with PRC2 and Exists as Short Fragments In Vivo

Previous RIP-seq analysis for the EZH2 subunit of PRC2 showed that reads derived from repetitive sequences comprised ˜20% of total reads—a not so insignificant fraction (Zhao et al., 2010)(FIG. 1A, right pie chart). We asked whether any family of repeat RNAs might be enriched relative to its representation in the transcriptome of female mouse embryonic stem (ES) cells, the cell type in which the RIP-seq analysis was performed. While most repeats were not enriched, we noted that SINEs accounted for ˜4% of all repetitive reads in the RIP-seq datasets and, within this family of repeats, the B2 element was enriched 4-fold above its representation in the female ES transcriptome (32% versus 8%; FIG. 1A) or the nuclear ES transcriptome (32% versus 12%, data from (Kung et al., 2015)). B2 RNA was highly enriched in RIP-seq reads relative to B1, another type of SINE repeat, in spite of the fact that the RNAs have similar expression profiles in the mouse genome (FIG. 1B, bottom panels)(Hasties, 1989). PRC2 therefore seems to have a preference for binding B2 RNA.

Examination of read distributions within the B2 element revealed an intriguing non-uniform pattern. Instead of the expected homogeneous distribution across the ˜200-nucleotide (nt) B2 element, we observed at least two subpopulations, with a sharp discontinuity of reads at ˜nt 98 (FIG. 1C). This pattern suggested that, apart from the full-length RNA, B2 may also exist as subfragments. The process of generating the RIP-seq libraries could have introduced biases in RNA fragmentation or cloning, however. Furthermore, only 36 bases could be sequenced by the older HiSeq2000 machine (Zhao et al., 2010). To rule out the possibility that the non-uniform RNA distributions arose from technical biases, we developed a short RNA-seq protocol that excludes an RNA fragmentation step and enriches for native transcripts in the 40- to 200-nt size range (see Experimental Procedures). Short RNA-seq of female mouse embryonic stem (ES) cells confirmed a discontinuity at nt 98 (FIG. 1D).

The discontinuity was interesting, as it occurred within the 51-nt critical region of B2 (nt 81-131; shaded region, FIG. 1E) previously shown by deletional analysis to be necessary and sufficient to stably bind an RNA docking site in POL-II in order to prevent formation of the pre-initiation complex (Espinoza et al., 2007; Ponicsan et al., 2015; Yakovchuk et al., 2009). To map the precise location of the break, we aligned 5′ ends of reads from the short RNA-seq library to the B2 consensus sequence and observed a strong peak at position 98 (FIG. 1E, “X”), with additional but smaller peaks at positions 77, 49, and 33. Thus, shorter forms of B2 RNA can indeed be detected in vivo.

To determine whether EZH2 binds B2 RNAs directly, we produced affinity-purified, recombinant EZH2 in baculovirus-infected insect cells and performed filter-binding assays with in vitro-transcribed B2 RNA. The results demonstrated that the full-length (180 nt) B2 RNA interacted with EZH2 and it did so with a dissociation constant (Ka) of 422.6±63 nM (FIG. 1F). It has an affinity that is similar to that of a similar-sized positive control, RepA I-II—a 210-nt shortened form of Xist RepA containing four of eight repeats (Cifuentes-Rojas et al. and FIG. 1F). This affinity was much greater than that for the negative control P4P6 RNA, a 154-nt transcript from Tetrahymena (K_(d)>3000 nM) and also for the 300-nt MBP RNA from E. coli. Truncating B2 RNA also resulted in extremely low affinities for EZH2, with various domains—DI [nt 1-72], DI+D2 [nt 1-105], and DIII [nt 99-140]—all demonstrating Ka of >3000 nM. These data demonstrate that B2 RNA directly interacts with EZH2 in vitro and confirm the binding interaction observed by RIP-seq in vivo.

Example 2. B2 RNA is Cleaved and Degraded in the Presence of EZH2

In principle, the discontinuity at position 98 could be due to an internal transcription start site or to an RNA processing event. Examination of the B2 sequence revealed internal Box A and Box B sites characteristic of RNA POL-III promoters and did not suggest additional transcription start sites around position 98 (FIG. 2A). Additionally, analysis of conventional and short RNA-seq data did not suggest a splice junction at position 98 or any other site of discontinuity. We therefore suspected a specific endonucleolytic event and set out to test this idea in vitro. Intriguingly, whereas incubation of 200 nM in-vitro-transcribed B2 RNA folded in 300 nM NaCl and supplemented with TAP100 buffer (incubation final concentrations: 5 nM Tris pH 7.9, 0.5 mM MgCl2, 0.02 mM EDTA, 0.01% NP40, 1% glycerol, 0.2 mM DTT) did not reveal any instability, addition of 25 nM purified recombinant PRC2 resulted in RNA fragmentation to sizes similar to those observed in vivo (FIG. 2B). This endonucleolytic event was recapitulated by addition of the EZH2 subunit alone, and was not observed with GST protein or with another PRC2 subunit, EED (FIG. 2C). We then performed deep sequencing of these RNA fragments to identify the exact cleavage sites. Several cleavage sites were observed, including a major one at position 98 and minor ones at positions 77 and 33 (FIG. 2D)—corresponding to the sharp discontinuities uncovered by EZH2 RIP-seq and the short RNA-seq analysis (FIG. 1C-E). Thus, the in vivo activity can be recapitulated in vitro using purified RNA and protein components (FIG. 2E). Collectively, these data demonstrate that full-length B2 RNA is subject to endonucleolytic cleavage at position 98, with minor cut sites at positions 77 and 33.

We next studied the in vitro kinetics of B2 RNA processing. In the presence of 25 nM EZH2, cleaved RNA accumulates over time between 0-100 minutes (FIG. 2F). To better understand the enhancement of B2 cleavage by EZH2, we plotted the amount of remaining full-length B2 RNA as a function of time (FIG. 2G). Cleavage rate constants were then determined by a linear fit using the differential form of the rate equation for an irreversible, first-order reaction (FIG. 2H). With either GST or no protein, we observed a low rate of turnover (k_(obs)=2×10⁻⁵ min⁻¹ and 6×10⁴ min⁻¹, respectively). The presence of EED mildly enhanced B2 cleavage at a modest rate of 8×10⁻³ min⁻¹. On the other hand, the presence of EZH2 resulted in a 1,400-fold rate increase to a k_(obs) of 0.029 (FIG. 2G)(R²>0.99, indicating that the datapoints have an excellent fit to the curve). Without EZH2, full-length B2 has an extrapolated half-life of 24 days in vitro. In the presence of EZH2, its half-life was reduced to 24 minutes (FIG. 2I). Thus, the ribonucleolytic cleavages within B2 are accelerated considerably by contact with PRC2.

The rate of cleavage also depended on EZH2 concentration. In the presence of 50 nM B2 RNA, increasingly higher processing rates were observed as the concentration of EZH2 was increased from 25 to 400 during a constant 20-minute incubation (FIG. 2H). Cleavage rate constants were again determined by fitting the data to a single-exponential function (FIG. 2J). At 25 nM EZH2, the observed rate constant, k_(obs), was 0.0248/min in the presence of 200 nM B2 RNA; at 125 nM EZH2, the k_(obs) was 0.2029/min; at 250 nM, the k_(obs) increased further to 0.3605/min; and at 500 nM EZH2, the k_(obs) still increased further to 0.4389 without reaching saturation (FIG. 2J-K). Taken together, the present data demonstrate that B2 RNA associates with PRC2 and induces a process that destabilizes B2 RNA, resulting in its cleavage into multiple fragments. These events occur both in vitro and in vivo.

Example 3. B2 RNA Induces Cell Death; Heat Shock Induces B2 Cleavage In Vivo

We asked whether degradation of B2 RNA is biologically relevant. First, we interrogated the consequences of introducing excess B2 RNA into NIH/3T3 cells, the cell line used previously to study B2 effects (Allen et al., 2004). Surprisingly, transfecting purified full-length B2 RNA into the cells resulted in marked cell death within 2 days of treatment (FIG. 3A). Culturing out to 3 days did not lead to cellular recovery. However, when B2 RNA was pre-incubated with EZH2 to induce cutting, cytotoxicity was reduced and cells grew to confluence within 3 days (FIG. 3A). We then repeated this analysis using a synthesized and purified truncated B2 fragment (nt 99-140), rather than one cut from full-length B2. Similar results were obtained: Starting with a transfection of 30,000 cells, full-length B2 RNA killed all cells within 2 days with no recovery after 5 days, whereas transfection of synthesized truncated B2 showed reduced cytotoxicity at 2 days and full recovery at 5 days (FIG. 3B). These data demonstrate that B2 RNA has biological activity in vivo and that cutting B2 RNA neutralizes that activity.

We set out to determine the nature of that activity. B2 RNA has been shown to block POL-II transcription during the heat shock response (Allen et al., 2004; Espinoza et al., 2004; Fornace and Mitchell, 1986; Li et al., 1999). Heat shock is a type of stress that puts cells at risk, and a rapid response is essential for survival (Chircop and Speidel, 2014). One immediate response is transcriptional downregulation of a large number of cellular genes—an adaptation to suppress expression of unnecessary genes. An equally critical immediate response is transcriptional upregulation of so-called “immediate early genes”. These genes are upregulated within the first 15 minutes after heat shock and encode proteins that buffer against cellular damage, such as those that assist in repair of damaged structures (FIG. 3C)(de Nadal et al., 2011). These proteins include transcription factors, epigenetic complexes, and chaperones that aid in refolding or elimination of damaged proteins. During the immediate early period, the B2 element is known to also increase in expression (Allen et al., 2004; Fornace and Mitchell, 1986).

To determine whether B2 RNA stability bears connection to heat shock, we examined the integrity of B2 RNA after 15 minutes of heat shock (45° C.) in NIH/3T3 cells. We performed short RNA-sequencing and compared the number of cut B2 fragments before and after heat shock. As B2 RNA levels also rose after heat shock (FIG. 3C), we normalized the number of cut sites to total B2 RNA levels in order to exclude increased B2 expression as a confounding factor. Intriguingly, a major increase in cutting was observed at position 98 after 15 minutes of heat shock, as well as at positions 77 and 33 (FIG. 3D). The difference in cutting before and after heat shock was highly significant (Kolmogorov-Smirnov [KS] test; P<0.0001). We conclude that B2 RNA has biological activity and temperature stress induces turnover of B2 RNA in vivo.

Example 4. B2 RNA Binds to Heat Shock-Responsive Genes

To understand the mechanism of action, we mapped genomic binding sites for B2 RNA using “capture hybridization analysis of RNA targets” with deep sequencing [CHART-seq (Simon, 2013; Simon et al., 2013)]. For capture probes, we designed complementary oligonucleotides to B2 RNA to pull down chromatin regions associated with B2 RNA. These 17-base capture probes spanned nt 87-103 of B2 RNA and overlapped the major cut site (FIG. 4A), thereby enabling us to specifically identify target sites bound by intact B2 RNA. Given variability of the B2 sequence, we designed a probe cocktail that would capture SNP variants for the vast majority of B2's in NIH/3T3 cells. CHART reads were then normalized to input DNA and to CHART reads obtained by a scrambled capture probe. Peaks were called using SICER (Xu et al., 2014) to identify statistically significant B2 targets sites throughout the genome (FDR<0.05). CHART-seq was conducted on pre- and post-heat shock cells (pre-H/S and post-H/S, respectively), and biological replicates showed highly similar results (FIG. 8).

Among 83,928 significant peaks altogether, 39,330 corresponded to nascent transcription from genomic B2 elements and served as positive controls (FIG. 8). Because the goal was to identify B2 target sites, peaks localizing within +/−3 kb of a B2 element (the average size of captured fragments) were excluded from further analysis. We examined the remaining 44,598 B2 RNA target sites. In pre-H/S cells, we observed 18,964 such sites. After only 15 minutes of heat shock, the number of B2 target sites nearly doubled to 31,368. Interestingly, target sites were largely non-overlapping between the two conditions. Among 18,964 pre-H/S sites, 13,230 were present only before heat shock (mentioned as “Type I” sites. Reciprocally, among 31,368 post-H/S sites, 25,634 were observed only after H/S (“Type II” sites). A minority (5,734) occurred in both pre- and post-H/S cells (“Type III” sites). We then characterized the target sites and found that the vast majority of B2 binding sites were in intergenic space and introns (FIG. 4B,C; pre-H/S shock shown), especially the first intron (FIG. 4B), and this was true for all three types of B2-binding targets (Tables S3-S5). With regards to the 1^(st) intron, the peaks often occurred at the 1^(st) exon-intron boundary and generally within 1,000 bp of the transcription start site (TSS), as shown by both a metagene analysis (FIG. 4D; KS test, P<0.0001) and by examination of specific genic loci (FIG. 4E). It should be emphasized, however, that B2 binding sites could occur anywhere within the gene body, that the binding sites tend to be broad and frequently spanned adjacent introns (FIG. 4E,S3), appearing different from the discrete peaks typified by transcription factors.

To determine how B2 binding affects gene expression, we performed RNA-seq analysis of NIH/3T3 cells before and after 15 minutes of heat shock and compared the results to B2 CHART-seq profiles. We observed that 1,587 genes were upregulated (log₂ fold-change≥0.5; Table 1) and 1,413 genes were downregulated (log ₂fold-change≤0.5; Table 2) by heat shock. Biological replicates were highly correlated and showed similar results (Pearson's R=0.9). Intriguingly, H/S-upregulated genes were enriched in the Type I subclass of B2 targets—i.e., they were bound by B2 RNA prior to heat shock, and were released from binding following heat shock. In contrast, H/S-downregulated genes were enriched in the Type II subclass—i.e., they were free of B2 binding prior to heat shock, but became B2 targets after heat shock. These trends are illustrated by specific examples (FIG. 4E). For instance, at two H/S-upregulated genes, A3galt2 and Snx32, B2 binding was observed in the resting state when the genes were expressed at low levels, but was lost after 15 minutes of heat shock after which the genes were upregulated. On the other hand, at two H/S-downregulated genes, Zfp37 and Zkscan5, B2 binding was not apparent before H/S, but became significant after H/S.

Metagene analysis confirmed these trends on a genome-wide scale (FIG. 4F,G). At 15 minutes post-H/S, the vast majority of genes displayed no changes in B2 localization (“all genes”). By contrast, H/S-upregulated genes (Table 1) showed a significant loss of B2 binding, and H/S-downregulated genes (Table 2) showed a significant increase in B2 binding. Together, these data demonstrate that B2 RNA targets specific genomic regions and that the binding pattern is rapidly and dramatically altered by heat shock. The changes are measurable within 15 minutes. We conclude that B2 RNA targets heat shock-responsive genes and its binding is anti-correlated with H/S gene expression across the genome.

Example 5. Cleavage of B2 RNA Induces Heat Shock-Responsive Genes

In light of the anti-correlation between B2 binding and target gene activity, the cleavability of B2 RNA raised a fascinating possibility: That B2 RNA might normally suppress POL-II activity, and that stress would trigger B2 turnover in order to lift the block to POL-II activity. To investigate this hypothesis, we performed ChIP-seq for the Serine-2 phosphorylated form of RNA POL-II (POL-II-S2P) to examine the density of elongating RNA polymerase across H/S-responsive genes (FIG. 5). As expected, genes upregulated by H/S (Table 1) showed increased POL-II density within 15 minutes of H/S, whereas genes downregulated by H/S (Table 2) showed decreased POL-II density (FIG. 5A, KS test, P<0.0001). We then examined the subset of genes that bind B2 only before H/S (Type I). Indeed, among the Type I genes, the H/S stimulus resulted in a significant spike in POL-II density (KS test; P<0.0001) (FIG. 5B,C), coinciding with the loss of the B2 binding (FIG. 4E,F). Conversely, among genes that bind B2 only after H/S (Type II), the H/S stimulus led to a significant decrease in POL-II density (KS test; P<0.0001) (FIG. 5B, C), coinciding with the gain of B2 binding within the same timeframe (FIG. 4E). Thus, POL-II activity is reduced where B2 binding appears, and POL-II activity increases where B2 binding is lost.

These data suggested that B2 binding is central to control of H/S genes. If so, turnover of B2 alone might be sufficient to induce transcriptional release. To de-couple B2 turnover from heat shock, we designed a B2-specific antisense oligonucleotide (ASO) using locked nucleic acid chemistry (LNA) to cleave B2 RNA. After 24 hours of transfection into NIH/3T3 cells (without heat shock), we observed significantly elevated cutting of B2 fragments relative to that seen in a scrambled (Scr) LNA-treated sample (FIG. 5D, KS test; P<0.0001). B2 LNA treatment recapitulated the increase in POL-II density across H/S-responsive genes, again without the heat shock stimulus (FIG. 5E, KS test; P<0.0001). Concurrently, RNA-seq analysis showed activation of H/S-responsive genes (FIG. 5F, KS test; P<0.0001). Biological replicates for RNA-seq and POL-II-S2P ChIP-seq showed excellent reproducibility. We conclude that increased POL-II density and gene expression can be uncoupled from the heat shock stimulus by ectopically inducing B2 degradation. Thus, B2 cleavage is central to the H/S response.

Example 6. EZH2 is Recruited to B2 Target Sites to Promote the Heat Shock Response

We were initially led to consider the role B2 RNA after noting its enriched representation in the EZH2 RIP-seq data (FIG. 1). We subsequently discovered that contact with EZH2 resulted in cleavage of B2 RNA in vitro (FIG. 2) and that cut forms of B2 RNA have dramatically reduced affinity for EZH2 (FIG. 1F). Together, these findings suggested that contact with EZH2 might destabilize B2 RNA and thereby release POL-II from suppression at H/S genes. To test this possibility, we performed EZH2 ChIP-seq in NIH/3T3 cells before and after heat shock and called statistically significant peaks of EZH2 enrichment using SICER (FDR<0.05), with biological replicates showing similar results. Consistent with EZH2's repressive role for transcription, we observed an enrichment for EZH2 at the TSS of H/S-downregulated genes (FIG. 6A). Unexpectedly, EZH2 also appeared to be slightly increased at H/S-upregulated genes, though this small increase was not statistically significant. However, the difference became pronounced and significant when analysis was focused on the subpopulation of H/S-upregulated genes bound by B2 (in the pre-H/S state) (FIG. 6B, KS test; P<0.0001). Those without a B2 site did not show increased EZH2 binding. Therefore, genes induced by heat shock paradoxically gained EZH2 coverage during activation. This finding implied that EZH2 is recruited to genes repressed by B2 RNA. Recruitment of EZH2 was not accompanied by an increase in trimethylation of H3K27, however (FIG. 6C). Rather, there was a decrease in H3K27me3 over the TSS after heat shock (FIG. 6D), consistent with their transcriptional upregulation.

Thus, during heat shock, EZH2 is recruited to inducible genes for a purpose other than H3K27 trimethylation. Because the paradoxical association between EZH2 density and gene expression was most remarkable for genic targets of B2 RNA (FIG. 6B) and in light of EZH2's effect on B2 RNA in vitro, we suspected that recruited EZH2 may serve to destabilize B2 RNA in order to activate target genes. Indeed, “meta-site” analysis from an EZH2-centric view (x=0 at EZH2 site) revealed that, after introduction of stress, EZH2 was attracted to sites where B2 was bound (FIG. 6E). In the converse analysis, a B2-centric view (x=0 at B2 sites) revealed the same finding—a gain of EZH2 binding where B2 binding was lost (FIG. 6F). This conclusion was supported by a very strong anti-correlation between change in B2 binding density and change in EZH2 coverage (FIG. 6G,H). Collectively, these data lend credence to the hypothesis that, in resting cells, B2 RNA is bound to H/S-inducible genes and a stressful stimulus triggers recruitment of EZH2, which in turn destabilizes B2 RNA for the activation of H/S genes.

Thus, EZH2 appears to play an equally important role in the heat shock response. We asked whether perturbing EZH2 affects B2 processing and gene induction in vivo. Administering ASOs specific for EZH2 to NIH/3T3 cells led to a significant knockdown (KD) of EZH2 (FIG. 9). Short RNA-seq analysis showed that this effect was accompanied by significantly decreased B2 cleavage at positions 98 and 77 (FIG. 61, KS test; P<0.0001). Depleting EZH2 also led to a blunted activation of H/S-responsive genes in two biological replicates (FIG. 6J, KS test; P<0.0001). These experiments thereby demonstrate that EZH2 is indeed a crucial factor in the induction of H/S-responsive genes.

The dynamic interplay between B2 RNA, EZH2, and POL-II activity can be appreciated by examination of specific H/S-inducible loci (FIG. 7A). For example, in resting cells, the gene for Cl tumor necrosis factor-related protein, Clqtnf3, was transcribed at low levels, as reflected by a low POL-II-S2P coverage (0.257) and a low RNA-seq value (FPKM=0.009). During rest, B2 RNA was bound at high levels and EZH2 binding was not detectable. Upon heat shock, EZH2 rapidly appeared within intron 3 at the same time that B2 binding decreased in introns 2 and 3. Concurrently, we observed increased POL-II-S2P coverage within the gene body (FPKM=0.308) and a 2.4-fold upregulation of CI qtnf3 transcription (FPKM=0.022). [N.B: The H/S genes respond in a graded rather than all-or-none manner (Brown et al., 1996; Chircop and Speidel, 2014; Kwak et al., 2013)]. Similarly, at another H/S-activated gene, Lrrc61, B2 binding disappeared when EZH2 binding appeared after heat shock, at which time POL-II-S2P coverage increased 2-fold (FPKM=0.093 to 0.192). All of these events were measurable within 15 minutes of heat shock. Ectopically cleaving B2 RNA (using B2 LNAs) recapitulated the H/S response in the absence of stimulus (FIG. 5E, 7A). For Clqtnf3, B2 LNA treatment resulted in a ˜2-fold increase of POL-II-S2P coverage (FPKM=0.481) and a 3-fold increase in RNA levels (FPKM=0.027) relative to baseline. For Lrrc61, there was a 2.3-fold increase of POL-II-S2 coverage (FPKM=0.213) and a 1.36-fold increase in transcription (FPKM=0.420) relative to baseline. We conclude that EZH2 and B2 play pivotal roles during the stress response, and that contact-induced B2 elimination is the key trigger for gene activation.

TABLE 1 List of heat shock-upregulated genes shown by RNA-seq analysis. Column A: Heat shock-upregulated gene shown by RNA-seq analysis of NIH/3T3 cells. Column B: Log2 fold-change of the gene in post-H/S cells relative to pre-H/S state. gene log2(fold_change) gene log2(fold_change) gene log2(fold_change) Pla2g4b 16.3925 Gimap9 2.97228 Cecr6 2.56515 H2-L 10.2141 Prr18 2.94858 Rps15a-ps4 2.54353 Rbm14-rbm4 9.70033 Col8a2 2.93686 Lyl1 2.53327 Btg3 7.92603 Slc10a1 2.90647 Gm10069 2.51012 Snora64 4.4165 Esr1 2.89912 5730480H06Rik 2.50066 Ccin 4.38112 Mfsd7c 2.89912 Il10 2.50066 Xrra1 4.10699 Muc1 2.89912 Lrrc4b 2.50066 H1fx 4.06394 Zfp72 2.89912 Mmp24 2.50066 Mc1r 3.80096 Cmah 2.84826 Snora44 2.50066 9630028B13Rik 3.72776 Cr2 2.8482 Tnfrsf13c 2.50066 Dusp18 3.68517 Klhl41 2.8482 Sap25 2.49155 Ctxn1 3.62625 Hspa1b 2.82076 2810442I21Rik 2.48463 Ism2 3.59641 Stxbp2 2.79653 4930565N06Rik 2.46801 Ipcef1 3.56099 Efnb3 2.78526 Col6a5 2.463 C1rb 3.53327 Actl7b 2.75266 Il1f9 2.463 Cyb561 3.50066 Snord15b 2.74752 Ppfibp2 2.4581 Camk4 3.49282 Gm17801 2.74178 1700020D05Rik 2.45648 Gpr1 3.46731 Gzmm 2.74178 Aldh1a3 2.43316 Doc2b 3.40167 Il17rb 2.74178 Gnat1 2.43316 Gpr3 3.39892 Tmem132b 2.74178 Nek10 2.43316 Pacsin1 3.2119 Hebp2 2.72196 Wnt6 2.43316 Rsph6a 3.19737 Xntrpc 2.68679 Rplp2-ps1 2.42774 A530013C23Rik 3.15779 BC055111 2.68487 Jam2 2.37402 Socs1 3.12905 Btbd18 2.68487 Olfr90 2.37402 Gm15107 3.12894 Fam219aos 2.68487 Gdap1 2.36384 Unc13d 3.12894 Fzd9 2.68487 Gpr82 2.36362 Zfp296 3.10073 Itga7 2.68487 Snora17 2.33896 1700001L05Rik 3.09658 Nwd1 2.68487 BC064078 2.33762 Upk1a 3.08563 1700113A16Rik 2.63311 Gm16287 2.33762 BC065397 3.08515 4930558J18Rik 2.62688 Tas1r1 2.33762 Jazf1 3.06248 Opn3 2.62688 Rnf43 2.32635 Ddn 3.04796 Wdr96 2.62688 Plxnc1 2.31983 Sh2d2a 3.0438 Gm10390 2.60575 Best1 2.29297 Bcl2l14 3.04098 Cxcl5 2.57392 Klhl40 2.29166 Brsk2 2.98871 Rbpjl 2.57344 Reep6 2.28961 6330403K07Rik 2.28945 A3galt2 2.05897 Wdr95 1.93686 Dqx1 2.28945 Mip 2.04852 Dpf3 1.93548 Gca 2.28945 Bhlhe41 2.04593 Snora21 1.91886 Gper1 2.28945 Trim72 2.04571 Pstpip1 1.91647 Jpx 2.28945 Igtp 2.04536 Sfrp5 1.9157 Trpt1 2.2764 Star 2.0433 Actr3b 1.90441 Sox15 2.25076 Fut2 2.0423 Hpgds 1.90441 Wdr78 2.24895 Plekha6 2.04223 Slfn8 1.90335 Msh4 2.24419 B430319G15Rik 2.04192 Hsph1 1.89988 Gm16702 2.23912 Gm3219 2.04192 Pdzd2 1.89678 Gbp3 2.23509 Kcnab3 2.04192 Mpeg1 1.87887 H2-Q1 2.21109 Pmel 2.04192 Dnajb1 1.87558 Cplx3 2.211 Tnni2 2.04192 Rhpn2 1.87141 E130310I04Rik 2.211 Gpr39 2.04098 Mgat4a 1.86854 Gnb3 2.211 Zpbp 2.04098 Ccdc166 1.84845 Homer2 2.211 Oas1b 2.04097 Slc1a2 1.84845 Nipal4 2.211 Opn1sw 2.02673 Al182371 1.84832 Serpina6 2.211 Fam221a 2.01216 1700112E06Rik 1.8482 Spata21 2.211 Fam83e 2.01138 1810010H24Rik 1.8482 Tas1r3 2.211 B3galt4 2.0113 B3gnt6 1.8482 Tppp 2.211 Snora26 2.00473 Coro2b 1.8482 Prickle3 2.20323 Kbtbd8 2.00401 Elfn1 1.8482 Adam1a 2.17995 Zfp783 2.00084 Gm3558 1.8482 Il18bp 2.17376 Gdf9 1.99587 Hsf5 1.8482 Ifitm5 2.16195 Gm12504 1.99377 Kcng4 1.8482 Dnah7b 2.15111 Raver2 1.99377 Myrf 1.8482 Stac3 2.15111 Klrg2 1.98508 Smim18 1.84815 Gm15760 2.15011 Nfe2l3 1.97729 Gm10941 1.8477 Snora24 2.14432 Masp2 1.95104 Phlda1 1.83422 Snora78 2.13742 Fcgbp 1.94831 Gm15545 1.83411 Gdpd1 2.12849 Gm6537 1.94831 4933413J09Rik 1.82881 Plcd4 2.1267 Gm6578 1.94831 Arhgef15 1.82881 Vmn1r58 2.11637 Med12l 1.94831 Cntn6 1.82881 Gm9159 2.10744 Serpinb1b 1.94831 Olfr1189 1.82881 Ccdc106 2.10658 Tmem82 1.94831 Rprl2 1.82509 Cers1 2.09773 Xylb 1.94831 Cep97 1.81814 Znf41-ps 2.09439 Hsf4 1.94331 Ddx60 1.81715 Cd68 2.09373 Slc6a20b 1.94114 LOC101669761 1.81715 Scn8a 2.09301 Kcnk7 1.9395 Klhdc9 1.81396 Vaultrc5 2.08566 Nacad 1.93879 1700022I11Rik 1.80786 Gt(ROSA)26Sor 2.07448 Ccpg1os 1.93686 Ttn 1.80658 Tha1 2.0712 Kcnh3 1.93686 Elmo3 1.80537 Rxfp3 1.79653 B3gnt5 1.685 Atp1a2 1.57835 Nipal1 1.79613 Glyctk 1.68497 Baiap2l1 1.57245 Mina 1.79225 Mboat1 1.68487 D3Ertd751e 1.57011 Tnfsf13 1.78146 Nodal 1.68487 Prdm9 1.5668 Rassf4 1.77813 Sh2d5 1.68487 Itih4 1.56527 Rdh9 1.77813 Myh7b 1.68431 1700034J05Rik 1.5652 Tlr1 1.77397 Dclre1c 1.67736 Raver1-fdx1l 1.56387 Ccdc28a 1.76904 Wnt2 1.67199 Tcf7 1.55402 Ccdc64b 1.76504 Gm16386 1.67005 Samd10 1.55366 Pde8b 1.7648 Lyn 1.66592 Celf3 1.55217 1110046J04Rik 1.75266 Phkg1 1.6648 Rel 1.55198 Cyp27b1 1.75266 Igfals 1.66368 Slc10a6 1.54397 Evpl 1.75266 2310014L17Rik 1.6616 Bend4 1.5425 Gm3230 1.75266 Nudt15 1.65918 Glp2r 1.54222 LOC102633315 1.75266 Pde1b 1.65858 Sptbn4 1.54168 Ppef1 1.75266 Pycard 1.64266 Rxfp4 1.54144 Csdc2 1.74675 Serpina3h 1.63923 Snhg10 1.54144 4930404N11Rik 1.74216 Nfam1 1.62808 Txlnb 1.54144 Gm11128 1.74178 Ptpro 1.62749 Hid1 1.53588 Lamc2 1.74178 Serpina1a 1.62696 Csf1r 1.53327 Lct 1.74178 Bspry 1.62688 Avpr2 1.53196 Ptgs2os 1.74178 Crabp2 1.62688 Qrfp 1.52681 Slc5a5 1.74178 Gm20756 1.62688 Gpd1 1.52636 Shank2 1.73997 Hcn3 1.62688 A330035P11Rik 1.51543 Gm13483 1.7356 Ptprcap 1.62688 Slc35g1 1.51543 Gpr61 1.72476 Rnf208 1.62688 Hspb6 1.50826 Prph 1.72265 Smok4a 1.62688 Ppfia3 1.50177 Pet117 1.72082 Unc13c 1.62688 G530011O06Rik 1.50141 Sema7a 1.7193 2900060B14Rik 1.6267 Papln 1.50105 1700003F12Rik 1.71621 Spta1 1.61997 Fmo5 1.50092 Tmem117 1.71621 Afap1l1 1.61903 Nr1h3 1.50072 Mtfr2 1.71536 Cldn3 1.61903 Ace2 1.50069 Nkpd1 1.7136 Nat8 1.61903 1700123M08Rik 1.50066 Loxl2 1.69758 Cul9 1.6178 4930592I03Rik 1.50066 Immp2l 1.69379 Dusp4 1.61584 6330403A02Rik 1.50066 Gng3 1.6927 Fcgr4 1.61584 A930007I19Rik 1.50066 Snora7a 1.68565 Gpr160 1.61512 Apol11b 1.50066 Liph 1.68547 Hspa1a 1.61007 Arhgef33 1.50066 4931403G20Rik 1.68527 Tnfrsf21 1.59665 Atcay 1.50066 Fam180a 1.68515 E330033B04Rik 1.59361 Ccdc121 1.50066 Gabre 1.68515 Zfp619 1.5887 Cldn22 1.50066 Gm5464 1.68515 Fbxl22 1.58506 Dpep2 1.50066 Gm4532 1.50066 Kndc1 1.42298 Zbtb46 1.33407 Gm7444 1.50066 D630041G03Rik 1.42212 Ugt1a7c 1.33286 Kbtbd11 1.50066 Lgals4 1.41708 Artn 1.33251 Klhl30 1.50066 Slc16a11 1.41091 Gdpd5 1.33179 Nat8l 1.50066 Gpr179 1.41042 Cd4 1.33151 Pih1d2 1.50066 Ranbp3l 1.40929 Ptplad2 1.33151 Prss27 1.50066 Amd2 1.40852 Wnt2b 1.32923 Prss8 1.50066 Pex1 1.40713 Hmga1-rs1 1.32721 Rsg1 1.50066 Plin4 1.40601 F2rl3 1.3266 Snora52 1.50066 Fbxo2 1.40581 Slc7a14 1.32262 Srrm3 1.50066 Trp53cor1 1.40434 4933421O10Rik 1.322 Infrsf11a 1.50066 Pde7b 1.39892 Gadd45b 1.32079 Zfp941 1.50066 Cntf 1.39812 4930562C15Rik 1.31983 Dlk2 1.49872 AK010878 1.39473 Map3k19 1.31983 Dmtn 1.49855 Trim68 1.39431 Map4k1 1.31983 Gm19705 1.49424 Htr2a 1.39336 2700054A10Rik 1.31827 Hoxc6 1.48708 Efcab4b 1.39168 Ttc38 1.31684 Col23a1 1.48359 Slc16a4 1.3892 BC068281 1.31495 Vipr1 1.48359 Snord22 1.38615 Dlgap2 1.31421 Gimap1 1.47889 Dph7 1.38372 Rhof 1.29987 Tmc4 1.47717 2210039B01Rik 1.38093 Snora74a 1.29838 Rdh12 1.4742 Gpr62 1.38093 Plekhg6 1.2981 Adcy7 1.47092 Slc23a1 1.38093 A930024E05Rik 1.29391 Ulk3 1.46879 Dper1 1.37458 Al317395 1.29391 Lag3 1.46553 Ttll13 1.37458 Eva1a 1.29391 1700007J10Rik 1.46387 Tctex1d4 1.37126 Snora28 1.29376 Kctd12b 1.463 Ccdc107 1.36936 5031414D18Rik 1.29297 Olfr1314 1.463 Ism1 1.36865 Ntrk3 1.28949 Slc25a18 1.463 Adam30 1.36334 Adamtsl1 1.28945 Zfp773 1.463 Tatdn3 1.35879 Esam 1.28945 Pianp 1.45782 D130040H23Rik 1.35337 Rltpr 1.28945 Msrb2 1.45731 Snora43 1.35152 Tmem240 1.28945 Tbc1d10c 1.45601 Ldb3 1.3509 Atf7ip2 1.28363 Prkd2 1.45336 Gpr173 1.34981 Amacr 1.2823 Rbmx2 1.45103 Mroh6 1.34981 Vegfb 1.27558 Arntl2 1.45055 Plce1 1.3453 Grip2 1.27432 Sycp2 1.44763 8430419L09Rik 1.34516 Slfn5 1.27378 Cdk5r1 1.44717 Bcl2l12 1.34451 Triqk 1.27105 Bag3 1.44633 4732491K20Rik 1.33815 Rpusd3 1.27038 Galc 1.44537 Duox1 1.33762 Ercc8 1.26377 Bcas3os1 1.43319 Ms4a6c 1.33762 Gm13826 1.26355 Pnma1 1.42344 Rtp4 1.33762 Kctd13 1.26328 BC051226 1.26309 Sobp 1.20643 Synpo2 1.15111 Podxl 1.26292 4933408B17Rik 1.20613 Alkbh7 1.14711 Slc35g3 1.26292 C030037D09Rik 1.20613 Tnik 1.14696 Hmgn5 1.25808 Tmem151a 1.20613 Slc16a6 1.1454 Cdt1 1.25486 Slc44a5 1.20547 Sema6b 1.14345 Kcnrg 1.25459 Fam189b 1.2043 C130083M11Rik 1.1432 Pcdhga4 1.25453 Gstp2 1.20335 Ppfia4 1.1432 Kcnma1 1.25351 Kcnc3 1.20036 Slc4a10 1.13882 Wnt4 1.25076 Rasl10a 1.20033 Pitpnm3 1.13748 9430091E24Rik 1.24955 C1qtnf3 1.20031 Macrod2 1.13675 Fam131a 1.24944 9030624G23Rik 1.2003 4930443O20Rik 1.13415 Kcnj15 1.24725 AY512931 1.2003 Khk 1.13195 Acyp2 1.24385 Adora2a 1.2003 Actr6 1.13087 Cenpv 1.24385 Cmya5 1.2003 Cspg5 1.12465 A930005H10Rik 1.24095 Gm16880 1.2003 Klhl36 1.12433 Abhd14a 1.24046 Gm8234 1.2003 Msantd1 1.12287 Naa30 1.23835 Nefh 1.2003 Epb4.1l5 1.11995 Zfp58 1.23825 Zglp1 1.2003 Grin3b 1.11589 Aamdc 1.23695 Slc25a14 1.1969 8430427H17Rik 1.11299 E330009J07Rik 1.23602 Ptgir 1.19468 Htr2b 1.11195 Lbp 1.23602 Map2k3 1.1943 Chrnb2 1.11104 Depdc7 1.23422 Ccdc101 1.19405 Al606473 1.11064 Celsr3 1.23292 Tinagl1 1.19082 Prorsd1 1.10873 Ociad2 1.23033 Serf1 1.1847 Slc26a6 1.10492 Napb 1.22772 Poc5 1.18338 Ufsp1 1.10078 Slc25a35 1.22618 Arid5a 1.18139 Kcnc1 1.10075 Nup210 1.22573 Col6a6 1.18093 Oip5 1.10073 Morn4 1.22452 Grpr 1.18014 Dnaic2 1.10063 Marveld3 1.22407 Ccl25 1.17567 Cdkn1c 1.10046 Zbtb3 1.21949 Fam96a 1.17447 2410004P03Rik 1.10043 Sphk1 1.21867 Zfp811 1.17432 Gngt2 1.10035 Nrip2 1.21705 Cdkl3 1.17415 1700020L24Rik 1.10019 Mapt 1.21439 Cecr2 1.17257 BC006965 1.10019 Acox2 1.2111 Smco4 1.17098 Dll1 1.10019 Cys1 1.21106 Pkp2 1.16547 Gm15455 1.10019 Actl10 1.211 Arc 1.16474 Tex38 1.10019 Ccdc40 1.211 Pcp4l1 1.16148 Lrriq3 1.10001 Clcn1 1.211 Cyp2d22 1.16078 Gbp10 1.09991 Mog 1.211 A230073K19Rik 1.15896 Grhl1 1.09522 Scube2 1.211 H2-T24 1.15681 Rab2b 1.09438 H2-T9 1.20949 Olfr543 1.15681 D8Ertd82e 1.09279 Rhbdl1 1.20785 Tmem40 1.15112 Foxl1 1.09279 Dedd2 1.09278 6030408B16Rik 1.04192 C1qtnf5 1.01755 Mtss1 1.08997 Arhgdig 1.04192 Gm10653 1.01638 Gm14446 1.08714 Cd74 1.04192 Cth 1.01507 Ppp1r3fos 1.08702 Ces1d 1.04192 Nrxn2 1.01162 Arl4d 1.08605 Gbx1 1.04192 Eif2d 1.01147 Pcdhga8 1.08499 Gm12522 1.04192 Rdh1 1.01121 Gm15645 1.08387 Gm6559 1.04192 Egr2 1.01105 Gpr21 1.08387 Gpbar1 1.04192 Herc3 1.01037 Tymp 1.08242 Gpr52 1.04192 Tmem251 1.00643 Cntn2 1.07872 Ifi205 1.04192 Angptl6 1.00496 Npr1 1.07872 L1cam 1.04192 Catsperg1 1.00496 Oas1c 1.07872 Lix1 1.04192 4833417C18Rik 1.00443 Olfm2 1.07872 Me3 1.04192 Cln3 1.0025 Zfp114 1.07872 Naaladl1 1.04192 Lingo2 0.997291 Angpt2 1.07759 Nap1l3 1.04192 Cyp2u1 0.994908 Gm5088 1.07699 Nlrp2 1.04192 Fam57a 0.994908 Klhl15 1.07463 Nmbr 1.04192 Trim7 0.994908 Dnajc17 1.07353 Npy1r 1.04192 Aipl1 0.993772 Foxo6 1.07299 Olfr267 1.04192 Kif27 0.993772 Prickle4 1.07299 Pkp1 1.04192 C130026I21Rik 0.991507 Setd4 1.07299 Rsl1 1.04192 Zscan29 0.987511 Snora70 1.07254 Serpinc1 1.04192 Vwa5b2 0.987476 Slc2a9 1.06974 Slc35g2 1.04192 Ldlrad4 0.98738 Slc4a11 1.06881 Sntb1 1.04192 Polr2d 0.985661 Surf2 1.06676 Tmem239 1.04192 Asxl3 0.984939 Mab21l3 1.0631 Tspan1 1.04192 Naip5 0.984869 Chd5 1.06304 Ccdc78 1.04189 Plin5 0.984791 4930488L21Rik 1.05868 Gnb5 1.04179 Cpeb2 0.98369 Pdzd7 1.05763 Cxx1b 1.04109 Gm1976 0.983577 1110008P14Rik 1.05495 Cd80 1.04098 Ptpre 0.983576 Snord15a 1.051 Gmpr2 1.03962 Pemt 0.983353 Al450353 1.05089 Snhg7 1.03798 Exd1 0.980189 Kdf1 1.04853 2310061I04Rik 1.03454 Vkorc1 0.978895 Msh5 1.04707 Gpt 1.03454 Tdg 0.978404 Tmem88 1.04701 Extl1 1.03012 Ecm2 0.978362 Atp6v0e2 1.04611 Nabp1 1.02862 Fuom 0.97786 Tgfb1 1.04536 Cd200 1.02751 Rnu12 0.977477 Nr4a2 1.04375 2810408I11Rik 1.02441 Zc2hc1c 0.976971 Snph 1.0423 Mapk10 1.02441 Unc119 0.976375 1700012D01Rik 1.04192 Gm7102 1.02141 Gm8801 0.975702 3632451O06Rik 1.04192 Gpr63 1.01962 Pdgfa 0.975251 4933406J10Rik 1.04192 Mcmdc2 1.01962 C2cd4c 0.973608 Tmem191c 0.972035 Ccdc73 0.917792 Rdh5 0.880761 Proser1 0.969728 Taf9b 0.917074 9130023H24Rik 0.88045 Ppapdc1b 0.969129 Prkaa2 0.917028 Cklf 0.880209 5730422E09Rik 0.968698 1110054M08Rik 0.917012 Apobec4 0.878874 Acyp1 0.966964 Zfp959 0.917012 Bai1 0.878874 Gprc5a 0.966757 Zfp595 0.916656 Ces1a 0.878874 Zfpm2 0.96574 C530005A16Rik 0.915701 Dusp23 0.878874 Ptprj 0.962058 Gm4432 0.915701 Gm20594 0.878874 Cpxm1 0.96165 Tnnt1 0.914369 Hal 0.878874 Slc25a16 0.958634 Cgref1 0.913576 LOC102634401 0.878874 9530027J09Rik 0.958632 Dancr 0.912835 Ppef2 0.878874 P2rx3 0.958372 Fastkd3 0.912483 Sycp3 0.878874 Spon1 0.957466 Slc8b1 0.911925 Ttc30a2 0.878874 Arntl 0.952404 Ttc39a 0.911876 Zfp459 0.878874 Bloc1s4 0.951861 Zbtb26 0.910565 Cdc25c 0.872104 Nfkbil1 0.951789 Osbpl10 0.907185 Akr1b3 0.871897 Tpcn1 0.95107 Adck3 0.907067 Notch3 0.871894 Camsap3 0.950006 Gm10578 0.906363 Tmem150b 0.871884 Gpm6b 0.948516 Itfg2 0.906018 Pde2a 0.87053 1700056E22Rik 0.948305 Megf11 0.905916 Ddx59 0.869902 Gabrb2 0.948305 Apol6 0.905812 Ggn 0.869005 Serac1 0.94768 3110040N11Rik 0.904664 Tysnd1 0.868374 Nckap5 0.946966 Dnaja4 0.903673 B930003M22Rik 0.867501 Fgd3 0.945867 Zmym1 0.903268 Cdcp1 0.867501 Rnd2 0.944931 Fahd2a 0.902592 Chst3 0.867501 Cyp4f13 0.943695 Plekhh1 0.902592 Rps6kl1 0.867501 Gramd1b 0.943094 Cdk20 0.900992 Zfp160 0.865721 Adam22 0.941898 Sbspon 0.899119 Pdf 0.865008 Tekt2 0.941898 Snord17 0.898693 Gm10845 0.864807 Scoc 0.941402 4930507D05Rik 0.898355 9330020H09Rik 0.864482 Slc39a6 0.939491 Zfp688 0.896366 Btbd6 0.86434 Ybey 0.938154 Sh2d4a 0.896038 Spef1 0.863728 Mtpap 0.936922 Slc7a11 0.893529 Dock8 0.862569 5730408K05Rik 0.934471 Pkn3 0.892733 Bdkrb1 0.86228 Xkr8 0.933427 D030028A08Rik 0.892603 Yy2 0.86228 Mtm1 0.933134 Al506816 0.892334 Hap1 0.860601 Porcn 0.932296 Tmem64 0.890878 Rrnad1 0.859938 Ugt1a6a 0.932118 Phyhd1 0.888334 Arl15 0.859273 1700094D03Rik 0.930346 Tpk1 0.887405 Pgap2 0.858987 Acsl6 0.927489 Nkiras1 0.884175 Cd302 0.857087 Agt 0.925678 Snora23 0.884144 Magohb 0.856945 Aurkaip1 0.922869 Lyrm2 0.8823 Thsd1 0.854136 Abcc6 0.853329 4933400F21Rik 0.846084 Mcts2 0.815087 Nnat 0.852521 Stau1 0.84469 Pim1 0.814866 Rps6ka1 0.848375 9030025P20Rik 0.844502 Gm12338 0.81463 Pex5l 0.848217 Lzic 0.84265 Mmachc 0.814113 Pla2g4c 0.848196 Paip1 0.842563 Endod1 0.814107 1700034I23Rik 0.848195 Fam213a 0.842291 Greb1l 0.813839 2510049J12Rik 0.848195 Gkap1 0.840528 Pam16 0.8135 6330418K02Rik 0.848195 Slc35b2 0.839747 Ncor2 0.812126 Adam1b 0.848195 4931440P22Rik 0.836634 Ap4e1 0.80971 Adrb3 0.848195 B630019K06Rik 0.835283 Nyap1 0.808223 Aldh3b2 0.848195 Prtg 0.832862 Mccc1os 0.805974 B130034C11Rik 0.848195 Pcdhga3 0.830016 Fam210b 0.805673 Bdkrb2 0.848195 Atxn3 0.829792 4933411K16Rik 0.805371 Cacna2d2 0.848195 Pms1 0.828572 Stab2 0.805371 Cacnb2 0.848195 Vamp1 0.828083 Tmem14c 0.805168 Ccdc170 0.848195 Dlg2 0.827534 Gfm2 0.80513 Cux2 0.848195 Nipal3 0.82751 Spaca6 0.80475 D730005E14Rik 0.848195 Ccrn4l 0.827023 Retn 0.803861 Ect2l 0.848195 Gm1943 0.826803 Nanos1 0.803319 Epsti1 0.848195 Mfsd8 0.826239 Dhrs13 0.802263 Fscn3 0.848195 Pfkp 0.825156 Rab7l1 0.802263 Ftcd 0.848195 Rprl3 0.824685 Fancg 0.801687 Gbp2b 0.848195 Al662270 0.824329 Jph3 0.799945 Gm10556 0.848195 Gpr151 0.824329 Zfp428 0.799896 Gm11149 0.848195 Osbpl6 0.82422 Uxt 0.796525 Gm11517 0.848195 Inhba 0.823205 Harbi1 0.796215 Gm15880 0.848195 Atpaf1 0.822957 Capns2 0.795969 Gm17746 0.848195 Cmc2 0.822775 Pabpc4l 0.795968 Gm4984 0.848195 Mrpl41 0.822763 Slc25a47 0.7942 Gpx3 0.848195 Relt 0.822405 Apip 0.793004 Itga4 0.848195 Sirt4 0.821295 Dbt 0.792254 Nkd2 0.848195 Snora81 0.82116 Rpph1 0.791102 Nupr1l 0.848195 Zfp846 0.820109 Jade3 0.790246 Olfr544 0.848195 Cmc1 0.818789 Alkbh2 0.789058 Panx3 0.848195 Kptn 0.817543 Cntd1 0.789058 Pde8a 0.848195 Leprotl1 0.817308 Fndc5 0.789058 Ppp1r3e 0.848195 Gna14 0.817146 Gm16982 0.789058 Srd5a2 0.848195 Fxyd1 0.81712 Slc24a5 0.789058 Wdfy4 0.848195 Mrpl1 0.816356 Tmem100 0.789058 Zfp85os 0.848195 Mob3b 0.815682 Zfp354b 0.789058 AU021063 0.848194 Commd4 0.815445 Zfp474 0.789058 Megf10 0.847764 Rmdn1 0.81537 Dpm2 0.789044 Igip 0.788349 6720416L17Rik 0.752659 Cbx7 0.739073 Vangl2 0.788187 Adcy5 0.752659 Chst12 0.739009 Mum1l1 0.787543 B3gnt3 0.752659 Alg13 0.738372 Adat3 0.785414 BC021767 0.752659 Plscr1 0.738264 2410018L13Rik 0.785263 Ccdc144b 0.752659 Gareml 0.737958 Gpr155 0.784518 Cldn15 0.752659 Morn1 0.737958 Mertk 0.783692 Ggt5 0.752659 Rfesd 0.736998 Tom1l1 0.781902 Gm10125 0.752659 Ago4 0.73664 Apbb1ip 0.780693 Gm10789 0.752659 Surf1 0.736503 Dennd1b 0.780558 Gm6251 0.752659 Urod 0.735173 Bbs4 0.779385 Kcnk3 0.752659 Vps8 0.735138 Fermt3 0.778882 Mslnl 0.752659 Tyw5 0.734593 Tmem161b 0.778178 Omp 0.752659 Trim34b 0.732648 Pex11a 0.778129 Rab26os 0.752659 Tssk6 0.732185 Shf 0.777706 Rab33a 0.752659 Ndufs6 0.731844 A130077B15Rik 0.773746 She 0.752659 Lrrc1 0.731533 4930455C13Rik 0.773479 Stmn1-rs1 0.752659 Exosc6 0.7314 Tmem128 0.771253 Stpg1 0.752659 Gpr4 0.731132 Ncf1 0.771184 Ttc25 0.752659 Eif5a2 0.730385 Flt3l 0.770416 Ccdc125 0.752641 Rnasek 0.72918 Timm21 0.770403 Nudt17 0.752439 Slc41a3 0.728341 Kif24 0.770009 Fahd1 0.752245 Hsp90aa1 0.727456 Foxj1 0.769525 Hvcn1 0.751942 Zfp524 0.727194 Trmt2b 0.768958 Tcp11l2 0.751931 Pogk 0.72698 Zfp558 0.768924 Cd320 0.74905 LOC106740 0.726647 C230091D08Rik 0.767682 Map3k13 0.749038 Stard5 0.726492 Trim59 0.764706 Phyhipl 0.747059 Prkar2b 0.726386 Ak6 0.763367 Dscc1 0.745278 Ttll3 0.72431 Lrrc61 0.761217 Mss51 0.745003 BC061194 0.724219 Slc25a27 0.760096 Camk2n2 0.744507 Nipa2 0.723398 Gm17762 0.759466 Asb3 0.743641 Zdhhc12 0.723354 Polq 0.75938 Emx2os 0.742987 Gm20319 0.722999 Apoo 0.757916 Depdc1a 0.742283 Gpcpd1 0.722965 Mrpl50 0.756048 Bok 0.741219 Col4a3bp 0.722612 Zfp874b 0.755962 Slc15a4 0.740891 Gnal 0.722065 Zfp954 0.755957 2610044O15Rik Arl6ip1 0.721104 Prss53 0.754948 8 0.740567 Snupn 0.72027 Peli3 0.754578 Mb21d2 0.740516 Sprtn 0.719836 Lfng 0.753516 Homer1 0.740491 Pnpo 0.718019 Pxdc1 0.753057 Prrg1 0.740343 Wdr8 0.71784 Phospho1 0.752661 Cnp 0.74021 Fbxo11 0.717345 4930539J05Rik 0.752659 Ramp2 0.740134 Cpne8 0.716441 Cpa4 0.716207 Gpr135 0.694222 Steap1 0.669469 Kcnj14 0.716207 Serpine1 0.692035 Cln6 0.66829 Ap3m2 0.714914 Slc38a9 0.689846 Tvp23b 0.667508 Bid 0.714076 Fcho2 0.689564 Hexdc 0.665967 Kri1 0.713345 Ints6 0.687629 Nr4a1 0.66566 Ankrd42 0.711881 Immp1l 0.687536 Pvt1 0.664854 Azin1 0.710642 Atg4d 0.687146 Mrpl32 0.664084 Pcdhac1 0.710402 Angpt1 0.685654 A230020J21Rik 0.663918 Ndufc1 0.709634 Begain 0.685588 Apol8 0.663706 Has3 0.709572 Pqlc2 0.685415 Gng8 0.663679 Aldh3b1 0.70932 Mfsd9 0.685326 Sdsl 0.663679 Shroom1 0.709143 1700120K04Rik 0.685153 Tmem223 0.663679 Awat2 0.707302 Cd14 0.684869 Clvs1 0.663678 Eps8l1 0.707095 Foxg1 0.683375 Apex1 0.661955 Smg9 0.706269 Ostm1 0.683047 Tmem192 0.661617 Gm8615 0.706096 Fbrs 0.68116 Siah1b 0.660784 Cgnl1 0.706094 Pqlc3 0.681088 Krcc1 0.65898 Dhx58 0.705249 Insig1 0.680904 Zeb2os 0.658912 Gm7609 0.704485 Lrch2 0.67938 Ahsa2 0.658866 Piga 0.702853 A230057D06Rik 0.678457 Aph1b 0.657954 Gpld1 0.702609 Sumo3 0.678457 Degs2 0.657643 Calcrl 0.701227 Tmem38b 0.678361 Pcdhga10 0.657617 Slc36a4 0.701085 Runx1 0.676638 Zfp329 0.657543 Tmem170b 0.700545 Efhc1 0.676024 9430038I01Rik 0.656592 Slc2a4rg-ps 0.70028 Parn 0.675847 Mfsd7a 0.656592 Ccdc53 0.700114 Fbxo41 0.675628 Tmem154 0.656592 Mns1 0.699875 Gba2 0.675114 Dtwd2 0.655861 Pyroxd1 0.699604 Ptrhd1 0.674611 Sla2 0.654917 Dcaf11 0.699481 Gng7 0.674239 Eef1e1 0.654614 Lrrtm2 0.699116 Mrpl15 0.67413 Nmral1 0.652852 Foxd2os 0.699048 Slc6a8 0.673973 Abcb9 0.651804 Tmem260 0.698446 Lmln 0.673293 Osbp 0.651032 Etohd2 0.697577 Ralgps2 0.673136 A730098P11Rik 0.650639 Smim13 0.696617 Rsph3b 0.672982 Pgbd5 0.648948 Vbp1 0.696407 Gm128 0.672774 Gpsm1 0.648374 Gm10033 0.696287 N6amt2 0.672643 Tbce 0.646814 Epha1 0.69572 Glrx3 0.672054 Mkl2 0.646378 Cd93 0.695059 Lyrm5 0.671061 Cep44 0.645635 Cradd 0.694944 Bckdhb 0.670957 Omd 0.645421 Zfyve19 0.694588 Ubxn2b 0.670957 Styx 0.643302 Lrrc73 0.694306 Tmem176b 0.670325 Klhl28 0.6429 Mettl22 0.694306 Strip2 0.670093 Rnf38 0.642831 Rad1 0.641986 Zfp300 0.623066 Nsg2 0.611039 Plekho2 0.641774 Adora2b 0.622927 Rab27b 0.610999 Rabl3 0.641702 Mnda 0.622737 Tmem258 0.610448 Pqlc1 0.640004 Tmem39a 0.622735 Smek1 0.609214 Katna1 0.639256 Gfpt2 0.622152 Olfm1 0.608263 Letm2 0.639051 Athl1 0.621666 Gprasp1 0.608247 Rpusd1 0.638148 Jmjd8 0.621474 Gm14005 0.608228 Mepce 0.637215 Pisd-ps3 0.621433 Isg15 0.606174 Prkra 0.636744 Cyb5rl 0.621432 Irgm1 0.605639 Zfp788 0.634809 2700046G09Rik 0.621166 Snhg4 0.605639 Fem1b 0.633894 Aox3 0.621166 Tst 0.605195 Ppm1h 0.633878 Gm2381 0.621166 Slc35e2 0.60484 Msl2 0.633096 Mmp16 0.621166 Ift20 0.604186 Chchd5 0.632246 Zfp273 0.621166 Ttc7b 0.603738 Irak4 0.631371 Fzd7 0.621147 Sirt5 0.603131 Slc43a2 0.631227 Thumpd2 0.621053 Dtymk 0.602386 Procr 0.630555 Phkg2 0.620933 Pdxp 0.601831 Peg3os 0.630487 Tmem181b-ps 0.620847 Wrap53 0.600599 Ece2 0.630018 Acad10 0.620113 Kdm4c 0.60056 Cdc42ep5 0.629752 Cckbr 0.61997 D430020J02Rik 0.599646 4933434E20Rik 0.629419 Fam151b 0.61997 Sft2d3 0.599477 Mif4gd 0.628942 Hpse 0.61997 Rnf19a 0.599175 Rsph3a 0.62888 Ptgdr2 0.61997 Zfp609 0.598705 C1galt1c1 0.628447 Lysmd2 0.619798 Apobec1 0.597047 Tmbim4 0.628297 Gsap 0.619637 Heca 0.597013 Cenph 0.628231 Ankrd39 0.619008 Sec61g 0.596673 Pecam1 0.627064 Ptges3l 0.618627 Tmem19 0.595994 BC028528 0.626879 Cbx4 0.618374 Psmg3 0.595282 C1ql3 0.626879 Lat2 0.617924 Zfp385c 0.594687 Ceacam16 0.626879 Ogfod3 0.617793 Cnih4 0.594569 Gm15408 0.626879 Gchfr 0.617511 Mppe1 0.594067 Fam198a 0.626827 Ube2q2 0.617001 Ten1 0.59351 Ift57 0.626357 Tac4 0.616837 Tmem200a 0.593417 Diexf 0.626265 Gm16023 0.616481 2010111I01Rik 0.593026 Lrrc39 0.626042 Mpc1 0.616368 Pisd-ps2 0.592919 Rnase10 0.626017 Tsg101 0.615968 Snx24 0.592641 Nlrp1b 0.624538 Wdr47 0.614698 Nfkbie 0.592566 Arxes1 0.62417 Pcnxl4 0.614302 5830415F09Rik 0.592261 Unc13b 0.623961 Klhl8 0.613586 Dcun1d2 0.592249 Hdac11 0.623461 Chek1 0.613071 Rgag4 0.591944 E230016K23Rik 0.623341 Chkb 0.612202 Dyx1c1 0.591382 Slc25a22 0.62327 Tmem126b 0.61188 Dcaf17 0.591272 Ciart 0.590954 Gtf2h4 0.574845 Tmem25 0.560892 Ramp3 0.590861 Ugt1a5 0.574447 Myo19 0.560238 Znrf2 0.589795 Lrrc8d 0.573823 Aif1l 0.559823 Mb21d1 0.58888 Zfp963 0.573677 Ppp2r5e 0.559413 Prkab2 0.58887 Prox2 0.573599 Scnm1 0.5593 Pla2g7 0.588629 Hoxd4 0.572448 Nomo1 0.558574 Efcab7 0.58861 Lig4 0.572442 Oma1 0.557833 B330016D10Rik 0.587808 Il17d 0.57166 Helq 0.557714 Kcnj13 0.587808 Ttpal 0.571422 Bivm 0.557124 A330009N23Rik 0.587798 Fam227a 0.57119 Caap1 0.556957 AK129341 0.58761 Tsc22d3 0.570947 Tgm4 0.556805 Agpat4 0.587377 Rnf111 0.570455 Mira 0.556405 Taf11 0.586982 Ube2m 0.57044 P2rx6 0.556297 Fst 0.58662 Abcd3 0.570293 Ap3s2 0.555981 Slc35f6 0.586565 Gab2 0.569957 Mettl10 0.555565 Cep70 0.585426 Casq1 0.568093 Perm1 0.555081 1110008F13Rik 0.583769 Gpr89 0.567585 Cdh18 0.554378 Acp6 0.582501 Dimt1 0.567419 3110002H16Rik 0.553881 Gtdc1 0.580918 Sccpdh 0.567194 Smpd5 0.55366 Klra2 0.57994 Ankrd9 0.566665 Pcdha10 0.553628 4833418N02Rik 0.57987 Polr2g 0.566507 Pms2 0.553541 Al848285 0.57987 Ap3m1 0.566406 Cyb5d2 0.553112 B130006D01Rik 0.57987 1500015A07Rik 0.566239 Exosc8 0.552342 C920025E04Rik 0.57987 5730508B09Rik 0.566151 Casz1 0.55191 Dusp3 0.579592 Chrm4 0.566151 Tmem107 0.551467 D930016D06Rik 0.578811 Plekhj1 0.565282 Chn1 0.551282 Ccdc84 0.578616 3110052M02Rik 0.564375 Dnal1 0.550887 A230103J11Rik 0.57856 Pkp3 0.564178 Ntn5 0.550711 Wdr89 0.578542 Arhgef39 0.564018 Rnd1 0.550337 Nav2 0.578471 Map3k8 0.563651 E530011L22Rik 0.550039 Dnah11 0.578348 Serinc4 0.56365 Slc9a3r2 0.549408 Ankle1 0.578103 Zfp345 0.563641 Gtf3c3 0.547369 Zkscan7 0.577966 Spopl 0.563258 Armc7 0.547319 Stx12 0.577634 Cdh24 0.563141 Tgfb3 0.547257 Cited1 0.577184 Ndfip2 0.562232 Tmem229b 0.546946 Wdr5b 0.576386 Pithd1 0.562121 Rgs16 0.545969 Mmadhc 0.576184 Osbp2 0.561933 Rfx3 0.545748 Sycp1 0.575501 Kin 0.561629 Dusp19 0.545573 Klf10 0.575321 Csnk2a2 0.561161 Cisd2 0.544746 A430078G23Rik 0.575229 Ccr9 0.561072 Gm20199 0.544746 Mdk 0.575135 Tmem184a 0.560921 Mfrp 0.544483 Pde4d 0.574991 Emid1 0.560892 3110062M04Rik 0.544427 Zfp446 0.544344 Col18a1 0.530327 Tmem183a 0.515984 Rnf13 0.544193 Aox1 0.529754 A830082K12Rik 0.515819 Styk1 0.543974 Camk1d 0.528434 Orai3 0.515617 Tyms 0.543539 Mrpl23 0.527912 Csmd3 0.515432 Npff 0.543132 Dph6 0.527526 Egf 0.515432 Trik1 0.542397 Cacng7 0.527458 Tmtc4 0.515432 Zdhhc4 0.542264 Zfp14 0.527404 Pcdhga6 0.514804 E030030I06Rik 0.541443 Cdc42se2 0.527181 Gm17066 0.514713 Fam228a 0.541443 2610002J02Rik 0.526737 Smim19 0.513809 Gm6583 0.541443 Hyls1 0.526574 Hist1h4i 0.513443 Zfp385a 0.540252 Tnni1 0.526432 Zfp935 0.513136 H2-K1 0.540178 Errfi1 0.526361 Gas5 0.513087 Stk19 0.540108 4930545L23Rik 0.526358 Serinc3 0.512927 Wdr55 0.539619 Clca1 0.526358 Trmt13 0.512829 1110001J03Rik 0.539364 Fscn2 0.526358 Mcts1 0.512614 Spred3 0.539216 Gm14379 0.526358 Zfp362 0.511695 Dpm3 0.53858 Mroh7 0.526358 Galnt13 0.511562 Tmem238 0.538559 Phf7 0.526358 Rce1 0.511331 Msrb1 0.538183 Zfp931 0.526358 Zufsp 0.511331 Psmd10 0.538183 Srpx2 0.526211 Ciita 0.511154 Tada3 0.538181 4833420G17Rik 0.526076 4921524J17Rik 0.510351 3110021N24Rik 0.537005 Creb3l1 0.525956 Fam92a 0.510289 Zfp174 0.536428 Rrp36 0.525482 Fam193b 0.509569 Zfp579 0.535497 Atg4b 0.524675 Adck5 0.509469 Atp6v1g2 0.534769 Hat1 0.524476 4930579G24Rik 0.509424 Icosl 0.534769 Cbfb 0.524265 Paqr3 0.509403 Tmem47 0.534065 Iba57 0.524034 Myom1 0.508284 Ube2b 0.533897 Pld1 0.523875 Tmem29 0.508004 Hscb 0.533385 Ehd4 0.523701 Dbhos 0.506884 Rb1 0.533144 Dram1 0.523638 Ntn1 0.506518 Slc45a3 0.533138 Mrps14 0.522991 Ap4s1 0.506184 Lamtor4 0.532759 Gp1ba 0.52285 Adprm 0.505908 Psmg1 0.532611 Fgfr3 0.522807 Vamp8 0.505153 Pigp 0.532384 Zfp1 0.521457 Ddt 0.504355 Gcnt7 0.532189 Sez6l2 0.52067 Stil 0.50419 Isg20 0.531979 Setd6 0.518642 Crtc3 0.503525 Grcc10 0.531928 Tnfsf12 0.517882 Pla2g12a 0.503497 Pi16 0.53156 Bbs10 0.517871 Naa38 0.503395 Usb1 0.53152 2700094K13Rik 0.517218 Nutf2-ps1 0.502546 2610301B20Rik 0.531452 Parpbp 0.5172 Polr1e 0.502282 Sh2d3c 0.530616 Qrsl1 0.516473 Slc52a2 0.501981 Tnr 0.530556 Acrbp 0.516059 Pcdhb22 0.50183 Gpatch3 0.501768 1700030J22Rik 0.500664 Serpina3g 0.500664 1700066M21Rik 0.501414 4930503E14Rik 0.500664 Slc40a1 0.500664 Bend6 0.501342 Alpk3 0.500664 Tmem204 0.500664 Ell2 0.501311 Gm13251 0.500664 Stox2 0.500652 Rbm7 0.50092 Gm6654 0.500664 Hoxa3 0.500622 Gulp1 0.500836 Ltc4s 0.500664 0610010B08Rik, 0.500664 Piwil2 0.500664 Gm4724 Rrad 0.500664

TABLE 2 List of heat shock-downregulated genes shown by RNA-seq analysis. Column A: Heat shock-downregulated gene shown by RNA-seq analysis of NIH/3T3 cells. Column B: Log2 fold−change of the gene in post-H/S cells relative to pre-H/S state. gene log2(fold_change) gene log2(fold_change) gene log2(fold_change) Ing4 −0.50031 Hs1bp3 −0.506507 Mpv17l −0.513925 Pcdhb2 −0.500595 Hist1h3c −0.506662 Zcchc3 −0.514007 Hist2h4 −0.500665 Zfp961 −0.507012 Mrpl22 −0.514214 Mef2c −0.501333 Ptpn6 −0.507148 Xist −0.514273 Bcdin3d −0.501479 Rdh13 −0.507474 Fam46b −0.514899 Hist3h2a −0.501508 Papolg −0.507547 Hist1h2ad −0.51514 Rnf32 −0.501903 Cpox −0.507785 Elavl2 −0.515613 Camkmt −0.502123 Nif3l1 −0.508042 Ino80c −0.515678 Mafg −0.502237 Dek −0.508221 Ccdc23 −0.516314 Leng1 −0.502735 Cmtm7 −0.509003 Eme1 −0.516865 Crnde −0.502792 Gm11974 −0.509778 Slc19a1 −0.517189 Scly −0.503023 Cyp4f16 −0.51008 Fam60a −0.517502 Enthd2 −0.503484 2210018M11Rik −0.510129 Zbtb24 −0.517857 Secisbp2 −0.503669 Jun −0.510715 Hemk1 −0.51791 Rbm20 −0.503733 Prr7 −0.510734 Glmn −0.518255 Creld2 −0.503796 Mllt6 −0.511174 2610020H08Rik −0.518407 Lcorl −0.503854 Shq1 −0.511474 Pcsk7 −0.518662 Rhpn1 −0.504378 4930577N17Rik −0.511662 Abtb1 −0.518668 A430005L14Rik −0.504389 Dna2 −0.511662 Ankrd6 −0.518812 Lace1 −0.504576 Tmem218 −0.512172 Rfxank −0.518862 Tmem208 −0.504576 Ppwd1 −0.512219 Zfp27 −0.518912 Fam50a −0.506063 Dbp −0.512643 Hist1h4b −0.518934 Irak3 −0.506246 Ip6k2 −0.513135 Naif1 −0.519346 Mamdc4 −0.506246 Prob1 −0.513266 Rab39b −0.519346 Mirg −0.51971 Pcca −0.533789 Siva1 −0.547497 Obscn −0.519902 Dnttip1 −0.533998 Zfp637 −0.547733 Slc4a1ap −0.519928 Birc2 −0.534003 Cry2 −0.548168 Pacsin3 −0.520093 Papd5 −0.534515 Bin3 −0.548322 Amn1 −0.520166 Prep −0.534706 0610009O20Rik −0.548329 Lrrc14b −0.520856 Gorasp1 −0.535042 3830408C21Rik −0.548597 Exosc4 −0.520914 Hist2h2ac −0.536325 Stk36 −0.549294 Mis18bp1 −0.521761 Ier2 −0.537189 Alkbh6 −0.549329 Hist1h2bf −0.522018 Nol12 −0.5375 Madd −0.54934 Jarid2 −0.522317 Mettl1 −0.537775 Tnfaip3 −0.549519 Ctgf −0.522406 Fgd6 −0.538283 Fbxl12 −0.549547 Zfp120 −0.522641 Ccne1 −0.538454 Thumpd1 −0.54967 Jph1 −0.524609 Mrpl42 −0.538658 Clcn6 −0.550539 Zfp93 −0.525308 Vmp1 −0.538673 4933411K20Rik −0.550935 Far2 −0.525753 2810021J22Rik −0.539498 Tmem129 −0.551641 Slc37a2 −0.525982 Tmem143 −0.539673 C330013E15Rik −0.552251 Slc7a7 −0.526089 Zkscan14 −0.539712 Zfp422 −0.552646 Coq7 −0.526739 Cdkn2d −0.539849 Dchs1 −0.553193 Epc1 −0.527036 Efcab11 −0.539849 Echdc1 −0.553488 Dhps −0.527047 A930013F10Rik −0.540539 Zfp775 −0.553516 Cbx8 −0.527184 Kif9 −0.540604 Scrn2 −0.553607 Hist1h2bn −0.527204 Uchl5 −0.540704 Rtkn2 −0.553639 N6amt1 −0.527226 Bmper −0.541647 Zfp90 −0.554355 Dguok −0.527277 AU040972 −0.543 Faim −0.554597 Nsun4 −0.527444 4930478L05Rik −0.543017 Slc25a29 −0.554769 Mob2 −0.527774 Agap3 −0.543024 Taf4b −0.555292 Ttc30b −0.528068 B230217C12Rik −0.543046 Psmc3ip −0.555487 Dpm1 −0.528659 Clca2 −0.543046 Ecsit −0.555716 Cd160 −0.528948 Efcab2 −0.543046 Cdk18 −0.555878 A130010J15Rik −0.529464 Fli1 −0.543046 Gm13212 −0.556088 Tex261 −0.529497 Adam33 −0.543153 Zfp809 −0.556774 Zrsr1 −0.529582 Zfp692 −0.543211 Slc27a6 −0.556931 Ezh2 −0.529736 Tmem37 −0.54398 Pagr1a −0.557216 Spns1 −0.529766 Exoc6 −0.543982 Ankrd61 −0.557364 Rad52 −0.530504 Nab1 −0.544948 2310061J03Rik −0.557451 A430105I19Rik −0.530628 Osgepl1 −0.545206 Atp5s −0.557451 D8Ertd738e −0.530884 Tdrp −0.54622 Taf6 −0.557831 Mettl23 −0.530933 Lzts1 −0.546333 BC005624 −0.558161 Hsdl2 −0.531341 Dtd1 −0.546666 Rpia −0.558475 Hmcn1 −0.532021 Sec23b −0.546755 Zfp110 −0.558722 C330018D20Rik −0.533362 Smg8 −0.54728 BC002163 −0.559052 Gzf1 −0.560191 B830017H08Rik −0.573752 Pnkp −0.587455 Ppp1r11 −0.560436 Cd55 −0.573752 Rgs4 −0.58759 Camta1 −0.560626 Cplx1 −0.573752 Ndufb2 −0.588812 Dennd6b −0.560699 D7Ertd715e −0.573752 Znrd1 −0.58887 Zfp958 −0.561342 E030018B13Rik −0.573752 Wdr76 −0.589025 Cog7 −0.561344 Frmd5 −0.573752 Tgif1 −0.589098 Slc35e4 −0.561346 Gm19466 −0.573752 Hist1h2bh −0.589503 Orc5 −0.562315 Itgb2 −0.573752 Srm −0.589822 Fam132b −0.562321 Mri1 −0.575174 1700037C18Rik −0.59005 Infrsf1b −0.562394 Terc −0.575417 Hmga2-ps1 −0.59005 Zfp551 −0.562656 Tacc2 −0.575468 Otud1 −0.590053 Zfp703 −0.563343 Gpr146 −0.575474 Klhl11 −0.590337 Tor4a −0.564252 Lgals6 −0.57582 Zfp606 −0.591307 Kcnk2 −0.564836 Ptpmt1 −0.576346 Il2rb −0.591498 Kctd19 −0.565341 Ngf −0.57681 Fam174a −0.592183 Zfp398 −0.565357 Mutyh −0.577625 Pacrgl −0.592657 Ift43 −0.565539 Wdr31 −0.577626 Gucd1 −0.593612 Arid3a −0.565912 Hinfp −0.577643 Zfp442 −0.594297 Klf11 −0.566662 Ppp1r13b −0.578079 Utp3 −0.595259 Ints5 −0.566901 Rgs19 −0.578324 Cdkn3 −0.595313 Ppapdc2 −0.567622 Jade2 −0.579041 Apcdd1 −0.595463 Tmed8 −0.567747 Hist1h1c −0.579818 Ccdc173 −0.595772 Spry2 −0.56794 Vsig10l −0.580002 Fam43a −0.596216 3830406C13Rik −0.568015 Sp110 −0.5801 Cir1 −0.596439 Dyrk2 −0.568265 Tcea2 −0.580364 Smn1 −0.596571 Cyp2j9 −0.569269 Tnfsf10 −0.580765 Ifi27l2a −0.596679 Ccdc55 −0.569922 Nt5m −0.581035 Siah1a −0.59683 Nat6 −0.570533 Mrps18b −0.581333 A330021E22Rik −0.597171 Haus4 −0.57081 Fgf18 −0.581553 Ppm1d −0.597613 Tmx2 −0.571123 Arhgap26 −0.582712 Zbtb39 −0.598211 Magee1 −0.571345 Brdt −0.582829 Fancf −0.598231 Urm1 −0.571663 Zfp169 −0.582877 Camk2b −0.59927 Zfp512 −0.571718 Egr3 −0.583242 Oard1 −0.599343 AU022252 −0.572398 Gatsl3 −0.583612 Cldn1 −0.599465 Zpr1 −0.572764 Tbc1d9 −0.584085 Npas2 −0.599465 Fam26e −0.572969 Magea8 −0.585681 Srp54b −0.599643 Tgds −0.57346 Tshz1 −0.58579 Zfp930 −0.6002 Hist1h2af −0.573751 Eed −0.586174 Rufy1 −0.601076 4930465K10Rik −0.573752 Prdm11 −0.586508 Mrpl54 −0.602695 4931431C16Rik −0.573752 Gm10336 −0.587345 Stx11 −0.602949 AA388235 −0.573752 Echdc3 −0.587408 Dusp6 −0.603491 Dnase1l1 −0.60358 Dda1 −0.623173 Ttc12 −0.636575 Gdnf −0.603686 Gcc1 −0.623266 Ypel4 −0.636706 Ldlrap1 −0.604216 Gdf5 −0.623313 Onecut2 −0.637626 B230319C09Rik −0.604244 Ap5b1 −0.623908 Polb −0.637657 Neu2 −0.60437 Ajuba −0.624013 Rhno1 −0.637914 Zfp839 −0.605325 Nek3 −0.624323 Eapp −0.640406 Apobr −0.605604 1700052N19Rik −0.624351 Gm20748 −0.64078 Gins3 −0.60594 Zc3h12b −0.624532 Mphosph10 −0.64086 H2afj −0.606179 Frg1 −0.624631 Zc3h3 −0.641326 Metap1d −0.606241 Sh3bp1 −0.62497 Abcd4 −0.641495 Rpap3 −0.606281 Sssca1 −0.625186 Stk35 −0.641874 Fbxo48 −0.607001 Arhgef19 −0.625299 Ccdc74a −0.643065 Scrn1 −0.607001 2610035D17Rik −0.625422 Pfkfb1 −0.643065 Zbtb8os −0.607287 Hps6 −0.626004 Ctbs −0.643279 Tgif2 −0.607855 C030039L03Rik −0.626041 Zfp84 −0.643772 Gstm4 −0.6093 Tstd3 −0.626207 Abt1 −0.64509 Tcn2 −0.609315 Zfyve21 −0.62677 Lpar6 −0.645267 Vps18 −0.609317 2810032G03Rik −0.627497 Mrpl44 −0.645493 Hist1h2bp −0.609375 Nfrkb −0.628125 Mapk1ip1 −0.645745 Oscp1 −0.610464 BC053749 −0.628174 Rfx5 −0.645847 Chst11 −0.610524 Fam161b −0.628174 Bsn −0.645863 Efna4 −0.610525 Dctd −0.628978 Chst1 −0.645863 Gm5069 −0.610917 Commd6 −0.629479 Mgst2 −0.645863 Kif3c −0.612129 Zfp59 −0.629547 Gm15401 −0.645877 Uap1l1 −0.612707 Edc3 −0.629571 Ptdss2 −0.64628 Slc16a2 −0.613014 Cecr5 −0.629599 Tmed1 −0.647055 Zfp960 −0.613692 Tprn −0.630454 Zbtb34 −0.648021 Hist1h3d −0.613986 Ccdc104 −0.630718 4930556M19Rik −0.648099 Itpk1 −0.614283 Ddx55 −0.631254 Ccdc174 −0.649049 Cdk6 −0.614877 Plod2 −0.632111 Krt10 −0.649049 Pex11g −0.614939 Fignl1 −0.632171 2810047C21Rik1 −0.649356 Arrdc4 −0.617362 Myo7a −0.633202 Dis3l2 −0.650614 Trp53rk −0.618256 2810408M09Rik −0.633783 Gpr75 −0.651521 2410004B18Rik −0.618544 Rad17 −0.634016 Necab3 −0.651521 Gins1 −0.619211 Rnf138 −0.634935 Dyrk3 −0.651559 Zfp532 −0.620083 Trim12c −0.635249 Snx11 −0.651727 Wnt10b −0.620199 Mettl15 −0.636089 Mid1ip1 −0.652493 Mr1 −0.620456 Hfe −0.636366 Rgs17 −0.652537 Zfp658 −0.620595 Fdxacb1 −0.636473 Zfp668 −0.654208 Ears2 −0.622258 Mrps28 −0.636473 Spock2 −0.693274 Loh12cr1 −0.622411 Fbxo32 −0.670724 Ttll11 −0.693274 Uhmk1 −0.654745 Cit −0.671092 5730507C01Rik −0.693346 Polr3a −0.655476 Slc16a9 −0.671699 Pibf1 −0.693752 Inca1 −0.655784 Snai2 −0.672634 Gm16596 −0.693878 Coq4 −0.655808 Zfp382 −0.672674 Lpin3 −0.694452 Ccnf −0.657503 Ifit1 −0.672916 Zfp341 −0.695049 4921513I03Rik −0.657561 Kcnj6 −0.673846 Trhde −0.697817 Fjx1 −0.657561 B4galt7 −0.674757 Haghl −0.69896 Gsg1l −0.657561 Il6ra −0.675251 Scx −0.699475 5830418K08Rik −0.657611 Lrrc48 −0.675405 Ankrd23 −0.699539 Tada2a −0.657686 Zc3hc1 −0.676349 Dok4 −0.699539 Zfp599 −0.658249 Trim21 −0.676785 Zfp759 −0.699539 A630066F11Rik −0.658756 Il34 −0.678002 Osr1 −0.700978 2210408I21Rik −0.659112 Zkscan5 −0.678454 Cxcl1 −0.701207 Rcan2 −0.659781 Fndc4 −0.679377 Capn5 −0.702153 Zfp248 −0.660258 Etohi1 −0.680126 Ftsj2 −0.702185 Nipsnap3b −0.661068 Nup210l −0.68017 Cbll1 −0.702813 Zfp947 −0.661354 Smim8 −0.68017 Trex1 −0.703789 Spryd7 −0.661689 Sharpin −0.680316 Terf1 −0.704221 1810043G02Rik −0.662097 Ddx27 −0.681203 Rsad1 −0.704583 4930453N24Rik −0.662222 Kctd21 −0.682037 Gla −0.705089 Armc8 −0.662384 Ifi44 −0.682371 Ccdc77 −0.705819 Tsen2 −0.66291 B4galt6 −0.682375 Eme2 −0.705906 Nhsl1 −0.663326 Pknox2 −0.683044 Tcf23 −0.70598 Dmnt3b −0.664391 Acy1 −0.683377 P2ry13 −0.706026 Hist1h2ai −0.664475 Dtnbp1 −0.683623 4933402D24Rik −0.706088 Apitd1 −0.664838 4931428F04Rik −0.685205 9530026P05Rik −0.706088 Itpkc −0.665082 Sema5a −0.685834 A330032B11Rik −0.706088 Foxf2 −0.665223 Mlycd −0.686426 Al854703 −0.706088 Plekha5 −0.666248 Bnc1 −0.686956 Aknad1 −0.706088 3110056K07Rik −0.666493 Hexim2 −0.687181 Apon −0.706088 Ftsj1 −0.666502 D330050I16Rik −0.688364 Aqp7 −0.706088 Slc39a8 −0.666549 Gltscr1 −0.688913 Cacna2d4 −0.706088 Primpol −0.66774 Lmf1 −0.689297 Dock3 −0.706088 2700069I18Rik −0.667935 Ubl3 −0.689301 Dusp15 −0.706088 Dffb −0.667935 Rnf220 −0.689847 Efcab8 −0.706088 Sgcd −0.667951 0610037L13Rik −0.690647 Fbxo47 −0.706088 Gm5512 −0.667976 Atl1 −0.691053 Gjb5 −0.706088 Mttp −0.668287 Tpgs1 −0.691596 Gm5779 −0.706088 Crebzf −0.669662 Sh3bp5 −0.692301 Gm6086 −0.706088 Pdik1l −0.670509 Csk −0.692498 Zbtb5 −0.728635 A430033K04Rik −0.670721 Gm20362 −0.708245 Mrps12 −0.729756 Gm9047 −0.706088 9230105E05Rik −0.709777 Deb1 −0.730002 Gpr84 −0.706088 Ikzf2 −0.710537 Pop7 −0.731847 Gstm7 −0.706088 Mxd3 −0.710562 Hoxa6 −0.732263 Hs3st6 −0.706088 Dlx1 −0.712027 Rnf113a2 −0.732286 Hsd17b14 −0.706088 Zfp873 −0.71301 A630072M18Rik −0.732412 Kif26a −0.706088 B9d1 −0.714355 Mocs3 −0.734491 Krt16 −0.706088 Esyt3 −0.71549 3830403N18Rik −0.736767 Pate2 −0.706088 Trit1 −0.716494 Cdrt4 −0.736767 Phyhip −0.706088 1810043H04Rik −0.718317 Hes3 −0.736767 Pld4 −0.706088 Hist1h2an −0.718552 Mmp28 −0.73698 Prss38 −0.706088 Lipt2 −0.718794 Pou5f2 −0.737044 Rag1 −0.706088 Gsdmd −0.719585 Card10 −0.737078 Rasgrp2 −0.706088 4921531C22Rik −0.720481 Lin37 −0.737085 Rbm3os −0.706088 Asic3 −0.720481 2010002M12Rik −0.737199 Rimbp3 −0.706088 Fkbpl −0.720481 Abhd15 −0.737199 Rnf183 −0.706088 Galr2 −0.720481 Gps2 −0.737236 Ryr3 −0.706088 Klf5 −0.720481 Hmga1 −0.738621 Slc17a9 −0.706088 Psmb9 −0.720481 Prpf38b −0.739789 Snora69 −0.706088 Tert −0.720481 Rfng −0.740839 Snord23 −0.706088 Rbm38 −0.720904 Il10rb −0.742049 Srpk3 −0.706088 Pot1b −0.72219 Dtd2 −0.742336 Tmem140 −0.706088 Lcmt1 −0.72227 Hsd3b7 −0.744176 Ttc24 −0.706088 Gtf3c6 −0.722322 Klc3 −0.745417 Tubg2 −0.706088 Cyb5d1 −0.723168 Pcdh10 −0.746236 Uchl4 −0.706088 Alkbh4 −0.723575 Mfap3l −0.751303 Unc45b −0.706088 Tmem205 −0.723904 Dnm3os −0.751652 Usp17la −0.706088 Foxc2 −0.724419 Pex7 −0.752295 Xkrx −0.706088 Slc2a8 −0.725225 Pgap3 −0.752692 Zfp389 −0.706088 Rin1 −0.725263 Phf11d −0.753085 Zim1 −0.706088 B4galnt2 −0.725681 Zfp189 −0.753931 2610203C22Rik −0.706095 Camk1g −0.725681 Smim1 −0.754017 Amy1 −0.706183 Ropn1l −0.725681 Adamts15 −0.754746 D630029K05Rik −0.706215 Zfp455 −0.725681 Mpp7 −0.755586 Crhr2 −0.706229 Fam83h −0.725929 Atg10 −0.75615 Tsen15 −0.706252 Sh3yl1 −0.7263 Nespas −0.75615 Tspan32 −0.706259 Lyrm1 −0.726373 Pctp −0.756909 5730420D15Rik −0.706377 Taf1c −0.727469 Pdlim1 −0.757538 Gcnt1 −0.706807 Irx1 −0.72786 Napepld −0.813393 Cntfr −0.706823 AW209491 −0.728344 Fbxw17 −0.81384 Fam206a −0.707078 Fbxo31 −0.72861 Ndufs5 −0.818532 Strada −0.707297 Slc8a2 −0.785323 Cyb561d1 −0.818996 Nanp −0.757989 Col11a2 −0.78544 Tlcd1 −0.819144 Zfp280b −0.759093 5930430L01Rik −0.786193 Plscr4 −0.819756 BC003965 −0.759857 Ganc −0.789766 Ndufaf1 −0.820008 Aaed1 −0.761054 Nxt2 −0.79019 1700029J07Rik −0.82059 Mrps9 −0.761167 Nfatc1 −0.790938 Abca8a −0.82059 Cenpn −0.761181 Mrps10 −0.791794 G6b −0.821414 Zfp748 −0.761254 Amt −0.795068 Oxsm −0.824032 Pcdhb19 −0.761436 Gm5577 −0.795068 Romo1 −0.824315 Plagl2 −0.762635 Zfp580 −0.795068 Tagap1 −0.824643 Stradb −0.76333 Det1 −0.795134 Ubac1 −0.826065 Tfap2a −0.763974 Ezh1 −0.795417 Stra13 −0.826126 Ugt1a6b −0.765062 2610305D13Rik −0.79557 Iqcd −0.826795 Rcor2 −0.765091 Ddx19a −0.795726 Unc5a −0.826795 Lactb −0.765161 Fam217b −0.795905 Nbn −0.830417 Emx2 −0.765782 Map3k5 −0.796144 Unc13a −0.831698 Haus1 −0.766594 Id1 −0.796365 Arhgap20os −0.832304 Gli2 −0.767552 Itgb4 −0.797031 Fam46c −0.832304 4930562F07Rik −0.76921 Irak1bp1 −0.798121 Gm4890 −0.83292 Kifc2 −0.76939 Hkdc1 −0.7997 Eno3 −0.833402 Gm6548 −0.770704 Pbld2 −0.7997 9630033F20Rik −0.833713 Gemin2 −0.770972 Zik1 −0.800245 Dpyd −0.834529 Plscr2 −0.771217 Mettl8 −0.802344 Fance −0.835103 Zfp418 −0.772085 Rab10os −0.803345 Gpr149 −0.8367 Pex12 −0.7726 Pias4 −0.804324 Kcnip2 −0.837577 Ankrd37 −0.773332 Fam188b −0.804752 BC039966 −0.837581 Ppargc1b −0.773872 Dnajb14 −0.80512 Fastkd1 −0.837581 Hes6 −0.775006 AW554918 −0.805369 Krt13 −0.837581 Vav3 −0.77609 Tigd3 −0.805577 Msl3l2 −0.837581 Mcur1 −0.778208 Rpl30 −0.807526 Neurl2 −0.837581 Fam216a −0.778209 Trappc5 −0.808742 Rarres2 −0.837581 Rhebl1 −0.779977 Rad9b −0.810325 Tdrd9 −0.837581 Snhg6 −0.780158 Gm3716 −0.810571 Zscan2 −0.837609 Zfp738 −0.780195 Shpk −0.810652 S100a13 −0.838208 Med27 −0.780297 Fam20a −0.810681 Cdca5 −0.840608 Gja1 −0.780555 Uqcc1 −0.81206 Ict1 −0.840648 Cstf1 −0.781231 Gm14139 −0.812224 Ggact −0.841342 Cxxc4 −0.781643 Gpr19 −0.812392 4930570G19Rik −0.841586 Mtus2 −0.782687 1600014C10Rik −0.812875 Kcnn1 −0.918356 Kiss1r −0.782897 Alg3 −0.812992 Rnpepl1 −0.918389 Saysd1 −0.784631 Atp10d −0.813057 Trmt5 −0.919185 Dusp2 −0.785323 Zfp764 −0.894501 Cryl1 −0.92023 Fignl2 −0.841642 Wdr44 −0.894865 Egfl6 −0.921283 E130307A14Rik −0.841942 Med26 −0.895078 Gm6402 −0.921283 Trim34a −0.842282 Zfp763 −0.896189 Hotair −0.921283 Pank1 −0.843037 Pusl1 −0.896236 Zfp708 −0.921564 Zfp191 −0.843053 Dgka −0.89726 Txnrd3 −0.923589 6430550D23Rik −0.84395 Yae1d1 −0.898458 Zan −0.936897 Syce2 −0.846123 2410076I21Rik −0.89981 Fam65b −0.936953 Nudt22 −0.846437 4930521E06Rik −0.89981 Parvb −0.937209 Rbm47 −0.847476 A330040F15Rik −0.89981 Pigw −0.940902 Irgm2 −0.847656 E130018N17Rik −0.89981 Lysmd4 −0.941065 Rft1 −0.849209 E430016F16Rik −0.89981 Zfp37 −0.941341 A330074K22Rik −0.849443 Fam184b −0.89981 Lekr1 −0.943815 1700029H14Rik −0.85006 Kctd4 −0.89981 Galnt9 −0.947365 Atp5sl −0.851423 Nipal2 −0.89981 Zfp943 −0.953224 Tmem14a −0.852202 Plekha7 −0.89981 Zfp87 −0.957457 As3mt −0.852315 Rims2 −0.89981 Gm12669 −0.958069 Mycn −0.852315 Soat2 −0.89981 1600029I14Rik −0.958083 Poli −0.85266 Hhatl −0.899876 2810405F15Rik −0.958083 Slc18a2 −0.854831 9230110C19Rik −0.902176 Aldh1l1 −0.958083 Rwdd2b −0.86081 Kbtbd4 −0.902319 Ap1g2 −0.958083 Rnase4 −0.865073 Tmem8 −0.902472 Bmp8b −0.958083 Epha7 −0.865657 Palb2 −0.903171 Camk2n1 −0.958083 Aqp11 −0.866944 Pard6a −0.904017 Ccdc87 −0.958083 Rep15 −0.866944 Nme3 −0.907648 Cd46 −0.958083 Grin2d −0.867395 C1qtnf1 −0.908103 Cml5 −0.958083 Gpr162 −0.868317 Frs3 −0.90817 Fxyd7 −0.958083 Dcbld1 −0.869465 Zmat1 −0.908467 Gm14057 −0.958083 Zfp597 −0.877144 Ap5s1 −0.910458 Gm6642 −0.958083 6330549D23Rik −0.877973 Zfp39 −0.910573 Kdm4d −0.958083 Gm10658 −0.878877 Zfp454 −0.911083 Tsacc −0.958083 Spata5l1 −0.878877 Gm10532 −0.912189 Uroc1 −0.958083 Arrb1 −0.87975 Dhx35 −0.912651 1810019D21Rik −0.958128 Acsf2 −0.882695 Hist1h1d −0.913021 Frs3os −0.958337 Hic2 −0.886541 Fosb −0.913754 Syt8 −0.959358 Nova2 −0.890182 Lrfn3 −0.913776 Kbtbd7 −0.961542 Gm7334 −0.890376 Zfp593 −0.914014 Rpusd2 −0.962275 Neat1 −0.890741 Lins −0.914152 Brms1 −0.962914 Mgmt −0.890925 Irx5 −0.915824 Eif1b −1.07147 Ankrd35 −0.891538 4930451G09Rik −0.916876 Mpp6 −1.07444 1700019G17Rik −0.892095 Klf2 −0.917442 Catip −1.07765 Atp6v0c-ps2 −0.893895 Tnfsf9 −1.02444 Drp2 −1.07888 Fam120aos −0.963613 Abhd1 −1.0251 Pcdhb8 −1.08078 Pfkfb4 −0.963796 Ccdc51 −1.02514 Bhlha15 −1.08206 Sv2a −0.963796 Srd5a1 −1.02627 Bricd5 −1.08206 Tmem185b −0.963796 Wdr53 −1.03014 Car15 −1.08206 1700086O06Rik −0.964385 Card14 −1.0313 Gm15612 −1.08206 Mitd1 −0.964645 Gm15446 −1.0313 Hspb9 −1.08206 Smco3 −0.964993 Gm6225 −1.0313 Rarb −1.08206 Col9a3 −0.965064 Krt80 −1.0313 Slc29a2 −1.08206 Tacr2 −0.968807 Sgpp2 −1.0313 Srcrb4d −1.08206 Tmem80 −0.973976 Trim36 −1.0313 Tubb4a −1.08206 Mcf2l −0.974236 Dolpp1 −1.03212 Gsto2 −1.08209 C4a −0.976222 Tmem220 −1.03226 Gmpr −1.08297 Zfp109 −0.980712 Gramd3 −1.0325 Zcchc5 −1.0843 Fam53b −0.981167 Plekha2 −1.03449 Pcdhgb8 −1.08517 4632427E13Rik −0.983515 Zfp108 −1.03621 Gm10509 −1.08634 Gm13157 −0.985491 Irf7 −1.03938 Gm17769 −1.08673 Akap5 −0.988789 1700021F05Rik −1.03988 Dbndd1 −1.08763 Gjb3 −0.988966 Map9 −1.04035 Katnal2 −1.0887 Pgbd1 −0.994904 B230217O12Rik −1.04191 Pip4k2a −1.08881 Fgfbp3 −0.996304 Col4a4 −1.04191 Mthfs −1.08891 Gm12070 −0.999898 Prr5l −1.04327 Casp4 −1.08983 Mir22hg −1.00059 Lrch4 −1.04389 9130019O22Rik −1.09251 Msi1 −1.0006 Snx32 −1.04743 Enpp3 −1.09271 3110009E18Rik −1.00099 Bcar3 −1.04746 8430431K14Rik −1.0935 Il15ra −1.00477 Commd9 −1.05007 Gm16712 −1.0935 9330151L19Rik −1.00508 Depdc1b −1.05105 Nuggc −1.0935 Adrb2 −1.00509 Pcdhga9 −1.05114 Dmkn −1.09763 Arhgef6 −1.00509 Zfp354a −1.05515 Bambi −1.09927 St6galnac2 −1.00509 Adhfe1 −1.0558 B4galnt4 −1.09955 A730017C20Rik −1.0051 Lcat −1.0586 Zfp677 −1.10137 Usp17le −1.00834 Pcdh12 −1.0586 Zfp870 −1.10137 Gan −1.01104 Slc44a3 −1.0586 Cmtr2 −1.10287 Ppdpf −1.01151 Rpp21 −1.06131 Mfsd6 −1.10351 Rassf7 −1.02042 Adamts13 −1.06243 Zfp408 −1.10399 Alyref2 −1.02132 Naf1 −1.06434 Mtap7d3 −1.10456 A630001G21Rik −1.0214 Clhc1 −1.06681 Nudt6 −1.11254 Zbtb49 −1.02217 Dhrs3 −1.06694 Larp6 −1.11285 Taf7 −1.02255 Trnau1ap −1.06825 Acn9 −1.29475 Ppm1e −1.02353 Ccdc64 −1.06964 Hist2h2ab −1.30242 Zfp30 −1.02424 Cdnf −1.06964 Cep41 −1.3043 Hist1h3g −1.02433 Gm10814 −1.18443 Pcdha12 −1.30484 Gpr85 −1.11496 Ccnj −1.18669 Cml1 −1.30544 9430018G01Rik −1.11501 Orai1 −1.18774 Zscan18 −1.31459 Gm14378 −1.11501 Cabyr −1.19303 Gpat2 −1.31476 Nmnat1 −1.11501 Sh3d21 −1.19876 Pkd2l2 −1.31833 Calml4 −1.1162 C030034I22Rik −1.19914 Nov −1.3192 Cyb561d2 −1.11762 Gm16740 −1.20283 Slc46a3 −1.32016 Hspa1l −1.12163 Crispld1 −1.20403 Rgs9bp −1.32674 Nupr1 −1.12472 Rap1gap −1.20765 Ap1s2 −1.33649 Zfp825 −1.13017 Nhej1 −1.21038 Mybl1 −1.33714 Rpp40 −1.13045 Apol9a −1.21719 Tusc1 −1.33963 Slc26a11 −1.1325 Kbtbd3 −1.22009 Mzf1 −1.34088 Trim65 −1.1325 Slc25a23 −1.22118 Zscan20 −1.34132 Ppargc1a −1.13279 Fbxl8 −1.22878 Tirap −1.34754 Tmem86a −1.13369 Hoxa1 −1.22939 Marveld2 −1.37816 Nudt16 −1.13415 Nat2 −1.23305 Akr1b10 −1.37926 Zfp202 −1.13696 Ndufaf6 −1.23343 Tulp2 −1.37931 Gdpgp1 −1.13954 Nlrc3 −1.23968 Omg −1.38002 Ccdc92 −1.14011 4931414P19Rik −1.24722 2300009A05Rik −1.38003 Pcdhgb4 −1.14036 Slc9a9 −1.24734 4933427E11Rik −1.38003 Thtpa −1.14065 Repin1 −1.24919 6230400D17Rik −1.38003 Tmtc1 −1.15184 Tspan2 −1.25039 Ankrd53 −1.38003 Mettl3 −1.15326 Btc −1.25262 Car5b −1.38003 Rab3a −1.15447 Spa17 −1.25262 Ccl9 −1.38003 C330006A16Rik −1.15655 Ccdc176 −1.25346 Cd247 −1.38003 Acvrl1 −1.15764 Raver1 −1.26039 E130102H24Rik −1.38003 Fancb −1.15797 2310068J16Rik −1.26102 Efcab5 −1.38003 Morn2 −1.15879 Dusp8 −1.26364 Epha10 −1.38003 Dusp14 −1.15914 Pidd1 −1.26865 Fam154b −1.38003 Naip6 −1.15914 Pgp −1.26976 Fer1l5 −1.38003 2010320M18Rik −1.16332 LOC100505025 −1.27565 Gm14634 −1.38003 4932416H05Rik −1.16416 Agpat2 −1.27578 Gm16523 −1.38003 Spdya −1.16524 Fpr1 −1.27578 Gm773 −1.38003 Srcin1 −1.16714 Gm20753 −1.27578 Igfbp2 −1.38003 Dlec1 −1.16812 F630042J09Rik −1.27804 Igflr1 −1.38003 Clcn2 −1.17179 Fam117a −1.28065 Lama5 −1.38003 Fam212a −1.17501 Ube2t −1.28523 Lect1 −1.38003 Myo1a −1.17567 A530032D15Rik −1.29105 Lenep −1.38003 Tubd1 −1.18154 Gm10791 −1.29105 2310009A05Rik −1.62251 Fam19a5 −1.18349 Gm6034 −1.29105 Gm15787 −1.62324 Acy3 −1.18443 Poln −1.29352 Ntf5 −1.62331 Lhx4 −1.38003 Cst6 −1.50625 Trpc2 −1.62464 Lrrc15 −1.38003 Ydjc −1.5258 Gm3435 −1.62687 Mroh8 −1.38003 Gm14124 −1.52882 Slc35d2 −1.6337 Nrg4 −1.38003 Zfp78 −1.53624 0610039K10Rik −1.64586 Rab20 −1.38003 Cideb −1.54305 Mettl20 −1.65482 Sag −1.38003 Col4a3 −1.54305 Pde3a −1.65756 Serpina3i −1.38003 E130012A19Rik −1.54305 Ccdc177 −1.6754 Spata20 −1.38003 E230008N13Rik −1.54305 Mterf1b −1.6754 Tmem144 −1.38003 Gm3604 −1.54305 Gm19557 −1.68489 Trcg1 −1.38003 Gpc3 −1.54305 Pde1a −1.68652 Zbtb32 −1.38003 Lrp2 −1.54305 Ccr7 −1.69782 Zfp750 −1.38003 Sh3tc1 −1.54305 Cdh22 −1.70609 2610027K06Rik −1.3801 Tex26 −1.54305 E230025N22Rik −1.70609 Cct6b −1.38046 Wnt8b −1.54305 Lypd1 −1.70609 Slx1b −1.39993 Emilin3 −1.54332 Olfr1417 −1.70609 Aph1c −1.4049 Abat −1.54336 Otoa −1.70609 Mapk11 −1.40895 Impg2 −1.54919 Pard3b −1.70609 Rnaset2a, −1.40933 Kcnh1 −1.54936 Ppm1j −1.70609 Rnaset2b Gimap6 −1.55225 Siglec15 −1.70609 Grk4 −1.42973 Il20rb −1.55225 St8sia1 −1.70609 4430402I18Rik −1.43644 Wdr93 −1.55225 Vmn2r-ps54 −1.70609 Foxd2 −1.44034 Gfi1 −1.55229 Col2a1 −1.70638 Mnd1 −1.44746 Tnfsf12Tnfsf13 −1.55406 Fam73a −1.70643 Phxr4 −1.45029 Lcmt2 −1.55828 Plekhg1 −1.70665 Hoxd3 −1.45722 Lsr −1.55834 Plb1 −1.70728 Spata24 −1.45823 1190005I06Rik −1.56266 Tenm2 −1.70774 Treml1 −1.46198 Gls2 −1.56293 Mis18a −1.71264 Gdap1l1 −1.46266 8430408G22Rik −1.5646 Pcbd2 −1.71272 Cpt1b −1.46299 Ppp1r3c −1.57178 Bbs5 −1.72048 Elovl4 −1.46384 3000002C10Rik −1.57375 Jph2 −1.73714 Ggct −1.46384 4930552P12Rik −1.57375 Cfp −1.7401 Tbx6 −1.46384 4931430N09Rik −1.57375 1700019L03Rik −1.74597 Zfp647 −1.46627 Prss12 −1.57375 Ushbp1 −1.74597 2410016O06Rik −1.46954 Gm2897 −1.57379 Dlgap1 −1.74779 Rpl14-ps1 −1.48126 Pcdhga2 −1.57681 Cobl −1.75624 G630090E17Rik −1.48442 Vash1 −1.58534 Siglec1 −1.76063 Svop −1.48477 Samd5 −1.58875 Cdh17 −1.76544 Tmem235 −1.48477 Fhl4 −1.59947 4930528A17Rik −1.77333 Ifitm1 −1.4849 2810008D09Rik −1.60233 Usp27x −2.19883 Leng9 −1.49253 Dand5 −1.60242 Ubald2 −2.22255 Slc25a2 −1.4971 Dnajc12 −1.61231 2310009B15Rik −2.25952 Gbp6 −1.77333 Gm10432 −1.95808 Stc2 −2.28001 2810410L24Rik −1.78516 Vmn1r43 −1.95808 Ppp1r1b −2.28554 Chrnb1 −1.78516 Scnn1a −1.96311 4930519F09Rik −2.29105 Kcnip3 −1.7866 Abhd3 −1.9638 Chn1os3 −2.29105 Cstad −1.80581 Gpr137c −1.96499 E130309D14Rik −2.29105 Rab27a −1.80581 Mapk12 −1.96499 Gsdmcl-ps −2.29105 Edaradd −1.82059 Itgae −1.96724 Zfp946 −2.31977 2700097O09Rik −1.82068 Zfp784 −1.99119 Frat1 −2.32787 Plp1 −1.8211 Fam195a −2.00996 Scd4 −2.32787 1810034E14Rik −1.83758 Plxdc1 −2.02214 Tex30 −2.32948 4933430I17Rik −1.83758 Rnasel −2.04804 Lincrna-cox2 −2.33623 Angptl7 −1.83758 Dtwd1 −2.05688 E2f2 −2.35593 BC039771 −1.83758 LOC100861615 −2.06437 Fam169b −2.38003 Ccdc38 −1.83758 3300002I08Rik −2.08206 Gm16062 −2.38003 Ccr10 −1.83758 Atg9b −2.08206 Nod2 −2.38003 Fam110c −1.83758 B3galt1 −2.08206 Usp13 −2.38003 Gata3 −1.83758 Ccdc17 −2.08206 12-Sep −2.42791 Glipr1 −1.83758 Foxq1 −2.08206 Ino80dos −2.44136 Npm2 −1.83758 Gnat2 −2.08206 Slc3a1 −2.46402 Rgag1 −1.83758 Krt83 −2.08206 1110019D14Rik −2.55225 Serpind1 −1.83758 Prlr −2.08206 B3gnt4 −2.55225 Gm16853 −1.83759 Zfp786 −2.08206 Ces4a −2.55225 Trim43c −1.8376 Gm19897 −2.08215 Dll4 −2.55225 Spns2 −1.83764 Aatk −2.08227 Usp18 −2.57375 4930506M07Rik −1.83767 9330159M07Rik −2.11376 C230029M16 −2.58557 Crmp1 −1.83774 1500011K16Rik −2.11501 Snrnp35 −2.59005 Fyb −1.83785 Mettl18 −2.1325 Edn1 −2.62687 Frem1 −1.87112 0610009L18Rik −2.13415 Luzp4 −2.62687 Grb14 −1.87888 2810002D19Rik −2.13415 Tssk2 −2.62687 Hspbap1 −1.8899 Anxa8 −2.15117 Mme −2.62733 Gm15987 −1.89981 Fsbp −2.15649 A530016L24Rik −2.65376 Lpcat2b −1.89981 1700024P16Rik −2.1728 Optc −2.65956 Neb −1.89981 Axin2 −2.18443 Cage1 −2.6754 Timp4 −1.89981 Ptprv −2.18443 Hpx −2.70609 Gm9855 −1.90588 Samd15 −2.18443 Armc2 −2.77333 Paqr7 −1.90629 Tmem252 −2.18443 Gm20257 −2.77333 Tmc3 −1.90629 1600020E01Rik −2.18457 Lmcd1 −2.77333 Tnfrsf14 −1.91198 Gm2373 −2.18509 Adcy3 −2.78028 Lhx6 −1.92398 Hdhd3 −2.1864 Pisd-ps1 −5.24468 Btbd8 −1.93985 Zfp472 −2.18696 Amd1, Amd2 −5.80573 Ttc30a1 −2.81623 Endog −3.0214 Raet1d −5.8346 Ccdc151 −2.82116 Itga10 −3.13415 Zfp91Cntf −7.85445 Ankdd1b −2.83758 Emc9 −3.18443 Gm20604 −11.0583 Atp8b4 −2.83758 D6Ertd527e −3.23872 Rsc1a1 −12.2259 Zfp712 −2.83758 Dmrta2 −3.28554 Mterf1a −2.87286 Gm14827 −3.33089 Sec1 −2.90629 Lrrc51 −3.86552 Tmem169 −2.96499 Jmjd7-pla2g4b −4.26272

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Other Embodiments

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. 

1.-15. (canceled)
 16. A composition comprising a plurality of isolated antisense oligonucleotides (ASOs), preferably each comprising at least one locked nucleotide, that target a plurality of different Alu or B2 sequences and mediate or promote cleavage of the sequences, and a pharmaceutically acceptable carrier. 17.-29. (canceled)
 30. A method of modulating health, proliferation potential, functionality or viability of a cell or tissue, the method comprising contacting the cell with an antisense oligonucleotide (ASO) comprising at least one locked nucleotide that binds to an Alu or B2 RNA and alters levels of the Alu or B2 RNA, by promoting or blocking cleavage of the B2/Alu RNA.
 31. The method of claim 30, wherein the cell is in a subject who suffers from an inflammatory or autoimmune disorder affecting the cell.
 32. The method of claim 30, wherein the cell is in a subject who suffers from a degenerative disorder affecting the cell.
 33. The method of claim 32, wherein the degenerative disorder is macular degeneration.
 34. A method of enhancing health or viability of a cell, the method comprising contacting the cell with an antisense oligonucleotide (ASO) comprising at least one locked nucleotide that binds to an Alu or B2 RNA and promotes cleavage of the Alu or B2 RNA, preferably wherein the ASO is an siRNA, shRNA or comprises at least one locked nucleotide.
 35. The method of claim 34, wherein the ASO is a gapmer or mixmer
 36. The method of claim 34, wherein the cell is in a subject who suffers from environmental stress such as infection, thermal, cold, radiation, hypoxic, and chemical exposure.
 37. The method of claim 36, wherein the cell is in a subject who suffers from an environmental stress.
 38. The method of claim 37, wherein the environmental stress is infection, thermal, radiation, or chemical exposure or hypoxic stress. 